FASMA: a service to format and analyze sequences in multiple alignments.

PubMed

Costantini, Susan; Colonna, Giovanni; Facchiano, Angelo M

2007-12-01

Multiple sequence alignments are successfully applied in many studies for under- standing the structural and functional relations among single nucleic acids and protein sequences as well as whole families. Because of the rapid growth of sequence databases, multiple sequence alignments can often be very large and difficult to visualize and analyze. We offer a new service aimed to visualize and analyze the multiple alignments obtained with different external algorithms, with new features useful for the comparison of the aligned sequences as well as for the creation of a final image of the alignment. The service is named FASMA and is available at http://bioinformatica.isa.cnr.it/FASMA/.

Prediction of β-turns in proteins from multiple alignment using neural network

PubMed Central

Kaur, Harpreet; Raghava, Gajendra Pal Singh

2003-01-01

A neural network-based method has been developed for the prediction of β-turns in proteins by using multiple sequence alignment. Two feed-forward back-propagation networks with a single hidden layer are used where the first-sequence structure network is trained with the multiple sequence alignment in the form of PSI-BLAST–generated position-specific scoring matrices. The initial predictions from the first network and PSIPRED-predicted secondary structure are used as input to the second structure-structure network to refine the predictions obtained from the first net. A significant improvement in prediction accuracy has been achieved by using evolutionary information contained in the multiple sequence alignment. The final network yields an overall prediction accuracy of 75.5% when tested by sevenfold cross-validation on a set of 426 nonhomologous protein chains. The corresponding Qpred, Qobs, and Matthews correlation coefficient values are 49.8%, 72.3%, and 0.43, respectively, and are the best among all the previously published β-turn prediction methods. The Web server BetaTPred2 (http://www.imtech.res.in/raghava/betatpred2/) has been developed based on this approach. PMID:12592033

A novel approach to multiple sequence alignment using hadoop data grids.

PubMed

Sudha Sadasivam, G; Baktavatchalam, G

2010-01-01

Multiple alignment of protein sequences helps to determine evolutionary linkage and to predict molecular structures. The factors to be considered while aligning multiple sequences are speed and accuracy of alignment. Although dynamic programming algorithms produce accurate alignments, they are computation intensive. In this paper we propose a time efficient approach to sequence alignment that also produces quality alignment. The dynamic nature of the algorithm coupled with data and computational parallelism of hadoop data grids improves the accuracy and speed of sequence alignment. The principle of block splitting in hadoop coupled with its scalability facilitates alignment of very large sequences.

SeqFIRE: a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments.

PubMed

Ajawatanawong, Pravech; Atkinson, Gemma C; Watson-Haigh, Nathan S; Mackenzie, Bryony; Baldauf, Sandra L

2012-07-01

Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.

Computational Framework for Prediction of Peptide Sequences That May Mediate Multiple Protein Interactions in Cancer-Associated Hub Proteins.

PubMed

Sarkar, Debasree; Patra, Piya; Ghosh, Abhirupa; Saha, Sudipto

2016-01-01

A considerable proportion of protein-protein interactions (PPIs) in the cell are estimated to be mediated by very short peptide segments that approximately conform to specific sequence patterns known as linear motifs (LMs), often present in the disordered regions in the eukaryotic proteins. These peptides have been found to interact with low affinity and are able bind to multiple interactors, thus playing an important role in the PPI networks involving date hubs. In this work, PPI data and de novo motif identification based method (MEME) were used to identify such peptides in three cancer-associated hub proteins-MYC, APC and MDM2. The peptides corresponding to the significant LMs identified for each hub protein were aligned, the overlapping regions across these peptides being termed as overlapping linear peptides (OLPs). These OLPs were thus predicted to be responsible for multiple PPIs of the corresponding hub proteins and a scoring system was developed to rank them. We predicted six OLPs in MYC and five OLPs in MDM2 that scored higher than OLP predictions from randomly generated protein sets. Two OLP sequences from the C-terminal of MYC were predicted to bind with FBXW7, component of an E3 ubiquitin-protein ligase complex involved in proteasomal degradation of MYC. Similarly, we identified peptides in the C-terminal of MDM2 interacting with FKBP3, which has a specific role in auto-ubiquitinylation of MDM2. The peptide sequences predicted in MYC and MDM2 look promising for designing orthosteric inhibitors against possible disease-associated PPIs. Since these OLPs can interact with other proteins as well, these inhibitors should be specific to the targeted interactor to prevent undesired side-effects. This computational framework has been designed to predict and rank the peptide regions that may mediate multiple PPIs and can be applied to other disease-associated date hub proteins for prediction of novel therapeutic targets of small molecule PPI modulators.

Embedding strategies for effective use of information from multiple sequence alignments.

PubMed Central

Henikoff, S.; Henikoff, J. G.

1997-01-01

We describe a new strategy for utilizing multiple sequence alignment information to detect distant relationships in searches of sequence databases. A single sequence representing a protein family is enriched by replacing conserved regions with position-specific scoring matrices (PSSMs) or consensus residues derived from multiple alignments of family members. In comprehensive tests of these and other family representations, PSSM-embedded queries produced the best results overall when used with a special version of the Smith-Waterman searching algorithm. Moreover, embedding consensus residues instead of PSSMs improved performance with readily available single sequence query searching programs, such as BLAST and FASTA. Embedding PSSMs or consensus residues into a representative sequence improves searching performance by extracting multiple alignment information from motif regions while retaining single sequence information where alignment is uncertain. PMID:9070452

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Bioinformatic prediction and in vivo validation of residue-residue interactions in human proteins

NASA Astrophysics Data System (ADS)

Jordan, Daniel; Davis, Erica; Katsanis, Nicholas; Sunyaev, Shamil

2014-03-01

Identifying residue-residue interactions in protein molecules is important for understanding both protein structure and function in the context of evolutionary dynamics and medical genetics. Such interactions can be difficult to predict using existing empirical or physical potentials, especially when residues are far from each other in sequence space. Using a multiple sequence alignment of 46 diverse vertebrate species we explore the space of allowed sequences for orthologous protein families. Amino acid changes that are known to damage protein function allow us to identify specific changes that are likely to have interacting partners. We fit the parameters of the continuous-time Markov process used in the alignment to conclude that these interactions are primarily pairwise, rather than higher order. Candidates for sites under pairwise epistasis are predicted, which can then be tested by experiment. We report the results of an initial round of in vivo experiments in a zebrafish model that verify the presence of multiple pairwise interactions predicted by our model. These experimentally validated interactions are novel, distant in sequence, and are not readily explained by known biochemical or biophysical features.

Classification of G-protein coupled receptors based on a rich generation of convolutional neural network, N-gram transformation and multiple sequence alignments.

PubMed

Li, Man; Ling, Cheng; Xu, Qi; Gao, Jingyang

2018-02-01

Sequence classification is crucial in predicting the function of newly discovered sequences. In recent years, the prediction of the incremental large-scale and diversity of sequences has heavily relied on the involvement of machine-learning algorithms. To improve prediction accuracy, these algorithms must confront the key challenge of extracting valuable features. In this work, we propose a feature-enhanced protein classification approach, considering the rich generation of multiple sequence alignment algorithms, N-gram probabilistic language model and the deep learning technique. The essence behind the proposed method is that if each group of sequences can be represented by one feature sequence, composed of homologous sites, there should be less loss when the sequence is rebuilt, when a more relevant sequence is added to the group. On the basis of this consideration, the prediction becomes whether a query sequence belonging to a group of sequences can be transferred to calculate the probability that the new feature sequence evolves from the original one. The proposed work focuses on the hierarchical classification of G-protein Coupled Receptors (GPCRs), which begins by extracting the feature sequences from the multiple sequence alignment results of the GPCRs sub-subfamilies. The N-gram model is then applied to construct the input vectors. Finally, these vectors are imported into a convolutional neural network to make a prediction. The experimental results elucidate that the proposed method provides significant performance improvements. The classification error rate of the proposed method is reduced by at least 4.67% (family level I) and 5.75% (family Level II), in comparison with the current state-of-the-art methods. The implementation program of the proposed work is freely available at: https://github.com/alanFchina/CNN .

MACSIMS : multiple alignment of complete sequences information management system

PubMed Central

Thompson, Julie D; Muller, Arnaud; Waterhouse, Andrew; Procter, Jim; Barton, Geoffrey J; Plewniak, Frédéric; Poch, Olivier

2006-01-01

Background In the post-genomic era, systems-level studies are being performed that seek to explain complex biological systems by integrating diverse resources from fields such as genomics, proteomics or transcriptomics. New information management systems are now needed for the collection, validation and analysis of the vast amount of heterogeneous data available. Multiple alignments of complete sequences provide an ideal environment for the integration of this information in the context of the protein family. Results MACSIMS is a multiple alignment-based information management program that combines the advantages of both knowledge-based and ab initio sequence analysis methods. Structural and functional information is retrieved automatically from the public databases. In the multiple alignment, homologous regions are identified and the retrieved data is evaluated and propagated from known to unknown sequences with these reliable regions. In a large-scale evaluation, the specificity of the propagated sequence features is estimated to be >99%, i.e. very few false positive predictions are made. MACSIMS is then used to characterise mutations in a test set of 100 proteins that are known to be involved in human genetic diseases. The number of sequence features associated with these proteins was increased by 60%, compared to the features available in the public databases. An XML format output file allows automatic parsing of the MACSIM results, while a graphical display using the JalView program allows manual analysis. Conclusion MACSIMS is a new information management system that incorporates detailed analyses of protein families at the structural, functional and evolutionary levels. MACSIMS thus provides a unique environment that facilitates knowledge extraction and the presentation of the most pertinent information to the biologist. A web server and the source code are available at . PMID:16792820

Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution

PubMed Central

Modahl, Cassandra M.; Mackessy, Stephen P.

2016-01-01

Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides access to cDNA sequences in the absence of living specimens, even from commercial venom sources, to evaluate important regional differences in venom composition and to study snake venom protein evolution. PMID:27280639

HIPPI: highly accurate protein family classification with ensembles of HMMs.

PubMed

Nguyen, Nam-Phuong; Nute, Michael; Mirarab, Siavash; Warnow, Tandy

2016-11-11

Given a new biological sequence, detecting membership in a known family is a basic step in many bioinformatics analyses, with applications to protein structure and function prediction and metagenomic taxon identification and abundance profiling, among others. Yet family identification of sequences that are distantly related to sequences in public databases or that are fragmentary remains one of the more difficult analytical problems in bioinformatics. We present a new technique for family identification called HIPPI (Hierarchical Profile Hidden Markov Models for Protein family Identification). HIPPI uses a novel technique to represent a multiple sequence alignment for a given protein family or superfamily by an ensemble of profile hidden Markov models computed using HMMER. An evaluation of HIPPI on the Pfam database shows that HIPPI has better overall precision and recall than blastp, HMMER, and pipelines based on HHsearch, and maintains good accuracy even for fragmentary query sequences and for protein families with low average pairwise sequence identity, both conditions where other methods degrade in accuracy. HIPPI provides accurate protein family identification and is robust to difficult model conditions. Our results, combined with observations from previous studies, show that ensembles of profile Hidden Markov models can better represent multiple sequence alignments than a single profile Hidden Markov model, and thus can improve downstream analyses for various bioinformatic tasks. Further research is needed to determine the best practices for building the ensemble of profile Hidden Markov models. HIPPI is available on GitHub at https://github.com/smirarab/sepp .

Sequence harmony: detecting functional specificity from alignments

PubMed Central

Feenstra, K. Anton; Pirovano, Walter; Krab, Klaas; Heringa, Jaap

2007-01-01

Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww. PMID:17584793

De novo identification of highly diverged protein repeats by probabilistic consistency.

PubMed

Biegert, A; Söding, J

2008-03-15

An estimated 25% of all eukaryotic proteins contain repeats, which underlines the importance of duplication for evolving new protein functions. Internal repeats often correspond to structural or functional units in proteins. Methods capable of identifying diverged repeated segments or domains at the sequence level can therefore assist in predicting domain structures, inferring hypotheses about function and mechanism, and investigating the evolution of proteins from smaller fragments. We present HHrepID, a method for the de novo identification of repeats in protein sequences. It is able to detect the sequence signature of structural repeats in many proteins that have not yet been known to possess internal sequence symmetry, such as outer membrane beta-barrels. HHrepID uses HMM-HMM comparison to exploit evolutionary information in the form of multiple sequence alignments of homologs. In contrast to a previous method, the new method (1) generates a multiple alignment of repeats; (2) utilizes the transitive nature of homology through a novel merging procedure with fully probabilistic treatment of alignments; (3) improves alignment quality through an algorithm that maximizes the expected accuracy; (4) is able to identify different kinds of repeats within complex architectures by a probabilistic domain boundary detection method and (5) improves sensitivity through a new approach to assess statistical significance. Server: http://toolkit.tuebingen.mpg.de/hhrepid; Executables: ftp://ftp.tuebingen.mpg.de/pub/protevo/HHrepID

Purification, developmental expression, and in silico characterization of α-amylase inhibitor from Echinochloa frumentacea.

PubMed

Panwar, Priyankar; Verma, A K; Dubey, Ashutosh

2018-05-01

Barnyard ( Echinochloa frumentacea ) and finger ( Eleusine coracana ) millet growing at northwestern Himalaya were explored for the α-amylase inhibitor (α-AI). The mature seeds of barnyard millet variety PRJ1 had maximum α-AI activity which increases in different developmental stage. α-AI was purified up to 22.25-fold from barnyard millet variety PRJ1. Semi-quantitative PCR of different developmental stages of barnyard millet seeds showed increased levels of the transcript from 7 to 28 days. Sequence analysis revealed that it contained 315 bp nucleotide which encodes 104 amino acid sequence with molecular weight 10.72 kDa. The predicted 3D structure of α-AI was 86.73% similar to a bifunctional inhibitor of ragi. In silico analysis of 71 α-AI protein sequences were carried out for biochemical features, homology search, multiple sequence alignment, phylogenetic tree construction, motif, and superfamily distribution of protein sequences. Analysis of multiple sequence alignment revealed the existence of conserved regions NPLP[S/G]CRWYVV[S/Q][Q/R]TCG[V/I] throughout sequences. Superfam analysis revealed that α-AI protein sequences were distributed among seven different superfamilies.

SVM-Prot 2016: A Web-Server for Machine Learning Prediction of Protein Functional Families from Sequence Irrespective of Similarity.

PubMed

Li, Ying Hong; Xu, Jing Yu; Tao, Lin; Li, Xiao Feng; Li, Shuang; Zeng, Xian; Chen, Shang Ying; Zhang, Peng; Qin, Chu; Zhang, Cheng; Chen, Zhe; Zhu, Feng; Chen, Yu Zong

2016-01-01

Knowledge of protein function is important for biological, medical and therapeutic studies, but many proteins are still unknown in function. There is a need for more improved functional prediction methods. Our SVM-Prot web-server employed a machine learning method for predicting protein functional families from protein sequences irrespective of similarity, which complemented those similarity-based and other methods in predicting diverse classes of proteins including the distantly-related proteins and homologous proteins of different functions. Since its publication in 2003, we made major improvements to SVM-Prot with (1) expanded coverage from 54 to 192 functional families, (2) more diverse protein descriptors protein representation, (3) improved predictive performances due to the use of more enriched training datasets and more variety of protein descriptors, (4) newly integrated BLAST analysis option for assessing proteins in the SVM-Prot predicted functional families that were similar in sequence to a query protein, and (5) newly added batch submission option for supporting the classification of multiple proteins. Moreover, 2 more machine learning approaches, K nearest neighbor and probabilistic neural networks, were added for facilitating collective assessment of protein functions by multiple methods. SVM-Prot can be accessed at http://bidd2.nus.edu.sg/cgi-bin/svmprot/svmprot.cgi.

Protein Sequence Classification with Improved Extreme Learning Machine Algorithms

PubMed Central

2014-01-01

Precisely classifying a protein sequence from a large biological protein sequences database plays an important role for developing competitive pharmacological products. Comparing the unseen sequence with all the identified protein sequences and returning the category index with the highest similarity scored protein, conventional methods are usually time-consuming. Therefore, it is urgent and necessary to build an efficient protein sequence classification system. In this paper, we study the performance of protein sequence classification using SLFNs. The recent efficient extreme learning machine (ELM) and its invariants are utilized as the training algorithms. The optimal pruned ELM is first employed for protein sequence classification in this paper. To further enhance the performance, the ensemble based SLFNs structure is constructed where multiple SLFNs with the same number of hidden nodes and the same activation function are used as ensembles. For each ensemble, the same training algorithm is adopted. The final category index is derived using the majority voting method. Two approaches, namely, the basic ELM and the OP-ELM, are adopted for the ensemble based SLFNs. The performance is analyzed and compared with several existing methods using datasets obtained from the Protein Information Resource center. The experimental results show the priority of the proposed algorithms. PMID:24795876

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

PubMed

Herrera, Victoria L M; Steffen, Martin; Moran, Ann Marie; Tan, Glaiza A; Pasion, Khristine A; Rivera, Keith; Pappin, Darryl J; Ruiz-Opazo, Nelson

2016-06-14

In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.

Design and construction of 2A peptide-linked multicistronic vectors.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Simple chained guide trees give high-quality protein multiple sequence alignments

PubMed Central

Boyce, Kieran; Sievers, Fabian; Higgins, Desmond G.

2014-01-01

Guide trees are used to decide the order of sequence alignment in the progressive multiple sequence alignment heuristic. These guide trees are often the limiting factor in making large alignments, and considerable effort has been expended over the years in making these quickly or accurately. In this article we show that, at least for protein families with large numbers of sequences that can be benchmarked with known structures, simple chained guide trees give the most accurate alignments. These also happen to be the fastest and simplest guide trees to construct, computationally. Such guide trees have a striking effect on the accuracy of alignments produced by some of the most widely used alignment packages. There is a marked increase in accuracy and a marked decrease in computational time, once the number of sequences goes much above a few hundred. This is true, even if the order of sequences in the guide tree is random. PMID:25002495

An ensemble approach for large-scale identification of protein-protein interactions using the alignments of multiple sequences

PubMed Central

Wang, Lei; You, Zhu-Hong; Chen, Xing; Li, Jian-Qiang; Yan, Xin; Zhang, Wei; Huang, Yu-An

2017-01-01

Protein–Protein Interactions (PPI) is not only the critical component of various biological processes in cells, but also the key to understand the mechanisms leading to healthy and diseased states in organisms. However, it is time-consuming and cost-intensive to identify the interactions among proteins using biological experiments. Hence, how to develop a more efficient computational method rapidly became an attractive topic in the post-genomic era. In this paper, we propose a novel method for inference of protein-protein interactions from protein amino acids sequences only. Specifically, protein amino acids sequence is firstly transformed into Position-Specific Scoring Matrix (PSSM) generated by multiple sequences alignments; then the Pseudo PSSM is used to extract feature descriptors. Finally, ensemble Rotation Forest (RF) learning system is trained to predict and recognize PPIs based solely on protein sequence feature. When performed the proposed method on the three benchmark data sets (Yeast, H. pylori, and independent dataset) for predicting PPIs, our method can achieve good average accuracies of 98.38%, 89.75%, and 96.25%, respectively. In order to further evaluate the prediction performance, we also compare the proposed method with other methods using same benchmark data sets. The experiment results demonstrate that the proposed method consistently outperforms other state-of-the-art method. Therefore, our method is effective and robust and can be taken as a useful tool in exploring and discovering new relationships between proteins. A web server is made publicly available at the URL http://202.119.201.126:8888/PsePSSM/ for academic use. PMID:28029645

GASP: Gapped Ancestral Sequence Prediction for proteins

PubMed Central

Edwards, Richard J; Shields, Denis C

2004-01-01

Background The prediction of ancestral protein sequences from multiple sequence alignments is useful for many bioinformatics analyses. Predicting ancestral sequences is not a simple procedure and relies on accurate alignments and phylogenies. Several algorithms exist based on Maximum Parsimony or Maximum Likelihood methods but many current implementations are unable to process residues with gaps, which may represent insertion/deletion (indel) events or sequence fragments. Results Here we present a new algorithm, GASP (Gapped Ancestral Sequence Prediction), for predicting ancestral sequences from phylogenetic trees and the corresponding multiple sequence alignments. Alignments may be of any size and contain gaps. GASP first assigns the positions of gaps in the phylogeny before using a likelihood-based approach centred on amino acid substitution matrices to assign ancestral amino acids. Important outgroup information is used by first working down from the tips of the tree to the root, using descendant data only to assign probabilities, and then working back up from the root to the tips using descendant and outgroup data to make predictions. GASP was tested on a number of simulated datasets based on real phylogenies. Prediction accuracy for ungapped data was similar to three alternative algorithms tested, with GASP performing better in some cases and worse in others. Adding simple insertions and deletions to the simulated data did not have a detrimental effect on GASP accuracy. Conclusions GASP (Gapped Ancestral Sequence Prediction) will predict ancestral sequences from multiple protein alignments of any size. Although not as accurate in all cases as some of the more sophisticated maximum likelihood approaches, it can process a wide range of input phylogenies and will predict ancestral sequences for gapped and ungapped residues alike. PMID:15350199

BlockLogo: visualization of peptide and sequence motif conservation

PubMed Central

Olsen, Lars Rønn; Kudahl, Ulrich Johan; Simon, Christian; Sun, Jing; Schönbach, Christian; Reinherz, Ellis L.; Zhang, Guang Lan; Brusic, Vladimir

2013-01-01

BlockLogo is a web-server application for visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms to enable on-the-fly prediction of MHC binding affinity to 15 common HLA class I and class II alleles as well as visual analysis of discontinuous epitopes from multiple sequence alignments. It enables the visualization and analysis of structural and functional motifs that are usually described as regular expressions. It provides a compact view of discontinuous motifs composed of distant positions within biological sequences. BlockLogo is available at: http://research4.dfci.harvard.edu/cvc/blocklogo/ and http://methilab.bu.edu/blocklogo/ PMID:24001880

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

PubMed Central

Laine, Elodie; Carbone, Alessandra

2015-01-01

Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

Effect of the sequence data deluge on the performance of methods for detecting protein functional residues.

PubMed

Garrido-Martín, Diego; Pazos, Florencio

2018-02-27

The exponential accumulation of new sequences in public databases is expected to improve the performance of all the approaches for predicting protein structural and functional features. Nevertheless, this was never assessed or quantified for some widely used methodologies, such as those aimed at detecting functional sites and functional subfamilies in protein multiple sequence alignments. Using raw protein sequences as only input, these approaches can detect fully conserved positions, as well as those with a family-dependent conservation pattern. Both types of residues are routinely used as predictors of functional sites and, consequently, understanding how the sequence content of the databases affects them is relevant and timely. In this work we evaluate how the growth and change with time in the content of sequence databases affect five sequence-based approaches for detecting functional sites and subfamilies. We do that by recreating historical versions of the multiple sequence alignments that would have been obtained in the past based on the database contents at different time points, covering a period of 20 years. Applying the methods to these historical alignments allows quantifying the temporal variation in their performance. Our results show that the number of families to which these methods can be applied sharply increases with time, while their ability to detect potentially functional residues remains almost constant. These results are informative for the methods' developers and final users, and may have implications in the design of new sequencing initiatives.

Prediction of G protein-coupled receptor encoding sequences from the synganglion transcriptome of the cattle tick, Rhipicephalus microplus

USDA-ARS?s Scientific Manuscript database

The cattle tick, Rhipicephalus (Boophilus) microplus, is a pest which causes multiple health complications in cattle. The G-protein coupled receptor (GPCR) super-family presents an interesting target for developing novel tick control methods. However, GPCRs share limited sequence similarity among or...

QuickProbs 2: Towards rapid construction of high-quality alignments of large protein families

PubMed Central

Gudyś, Adam; Deorowicz, Sebastian

2017-01-01

The ever-increasing size of sequence databases caused by the development of high throughput sequencing, poses to multiple alignment algorithms one of the greatest challenges yet. As we show, well-established techniques employed for increasing alignment quality, i.e., refinement and consistency, are ineffective when large protein families are investigated. We present QuickProbs 2, an algorithm for multiple sequence alignment. Based on probabilistic models, equipped with novel column-oriented refinement and selective consistency, it offers outstanding accuracy. When analysing hundreds of sequences, Quick-Probs 2 is noticeably better than ClustalΩ and MAFFT, the previous leaders for processing numerous protein families. In the case of smaller sets, for which consistency-based methods are the best performing, QuickProbs 2 is also superior to the competitors. Due to low computational requirements of selective consistency and utilization of massively parallel architectures, presented algorithm has similar execution times to ClustalΩ, and is orders of magnitude faster than full consistency approaches, like MSAProbs or PicXAA. All these make QuickProbs 2 an excellent tool for aligning families ranging from few, to hundreds of proteins. PMID:28139687

Characterization of tannase protein sequences of bacteria and fungi: an in silico study.

PubMed

Banerjee, Amrita; Jana, Arijit; Pati, Bikash R; Mondal, Keshab C; Das Mohapatra, Pradeep K

2012-04-01

The tannase protein sequences of 149 bacteria and 36 fungi were retrieved from NCBI database. Among them only 77 bacterial and 31 fungal tannase sequences were taken which have different amino acid compositions. These sequences were analysed for different physical and chemical properties, superfamily search, multiple sequence alignment, phylogenetic tree construction and motif finding to find out the functional motif and the evolutionary relationship among them. The superfamily search for these tannase exposed the occurrence of proline iminopeptidase-like, biotin biosynthesis protein BioH, O-acetyltransferase, carboxylesterase/thioesterase 1, carbon-carbon bond hydrolase, haloperoxidase, prolyl oligopeptidase, C-terminal domain and mycobacterial antigens families and alpha/beta hydrolase superfamily. Some bacterial and fungal sequence showed similarity with different families individually. The multiple sequence alignment of these tannase protein sequences showed conserved regions at different stretches with maximum homology from amino acid residues 389-469 and 482-523 which could be used for designing degenerate primers or probes specific for tannase producing bacterial and fungal species. Phylogenetic tree showed two different clusters; one has only bacteria and another have both fungi and bacteria showing some relationship between these different genera. Although in second cluster near about all fungal species were found together in a corner which indicates the sequence level similarity among fungal genera. The distributions of fourteen motifs analysis revealed Motif 1 with a signature amino acid sequence of 29 amino acids, i.e. GCSTGGREALKQAQRWPHDYDGIIANNPA, was uniformly observed in 83.3 % of studied tannase sequences representing its participation with the structure and enzymatic function.

A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene.

PubMed Central

Walker, M D; Park, C W; Rosen, A; Aronheim, A

1990-01-01

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on multiple DNA sequence elements located in the 5' flanking region of the gene. Of particular importance in the rat insulin I gene are two closely similar 9 bp sequences (IEB1 and IEB2): mutation of either of these leads to 5-10 fold reduction in transcriptional activity. We have screened an expression cDNA library derived from mouse pancreatic endocrine beta cells with a radioactive DNA probe containing multiple copies of the IEB1 sequence. A cDNA clone (A1) isolated by this procedure encodes a protein which shows efficient binding to the IEB1 probe, but much weaker binding to either an unrelated DNA probe or to a probe bearing a single base pair insertion within the recognition sequence. DNA sequence analysis indicates a protein belonging to the helix-loop-helix family of DNA-binding proteins. The ability of the protein encoded by clone A1 to recognize a number of wild type and mutant DNA sequences correlates closely with the ability of each sequence element to support transcription in vivo in the context of the insulin 5' flanking DNA. We conclude that the isolated cDNA may encode a transcription factor that participates in control of insulin gene expression. Images PMID:2181401

BLAST and FASTA similarity searching for multiple sequence alignment.

PubMed

Pearson, William R

2014-01-01

BLAST, FASTA, and other similarity searching programs seek to identify homologous proteins and DNA sequences based on excess sequence similarity. If two sequences share much more similarity than expected by chance, the simplest explanation for the excess similarity is common ancestry-homology. The most effective similarity searches compare protein sequences, rather than DNA sequences, for sequences that encode proteins, and use expectation values, rather than percent identity, to infer homology. The BLAST and FASTA packages of sequence comparison programs provide programs for comparing protein and DNA sequences to protein databases (the most sensitive searches). Protein and translated-DNA comparisons to protein databases routinely allow evolutionary look back times from 1 to 2 billion years; DNA:DNA searches are 5-10-fold less sensitive. BLAST and FASTA can be run on popular web sites, but can also be downloaded and installed on local computers. With local installation, target databases can be customized for the sequence data being characterized. With today's very large protein databases, search sensitivity can also be improved by searching smaller comprehensive databases, for example, a complete protein set from an evolutionarily neighboring model organism. By default, BLAST and FASTA use scoring strategies target for distant evolutionary relationships; for comparisons involving short domains or queries, or searches that seek relatively close homologs (e.g. mouse-human), shallower scoring matrices will be more effective. Both BLAST and FASTA provide very accurate statistical estimates, which can be used to reliably identify protein sequences that diverged more than 2 billion years ago.

ComplexContact: a web server for inter-protein contact prediction using deep learning.

PubMed

Zeng, Hong; Wang, Sheng; Zhou, Tianming; Zhao, Feifeng; Li, Xiufeng; Wu, Qing; Xu, Jinbo

2018-05-22

ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Prediction of protein secondary structure content for the twilight zone sequences.

PubMed

Homaeian, Leila; Kurgan, Lukasz A; Ruan, Jishou; Cios, Krzysztof J; Chen, Ke

2007-11-15

Secondary protein structure carries information about local structural arrangements, which include three major conformations: alpha-helices, beta-strands, and coils. Significant majority of successful methods for prediction of the secondary structure is based on multiple sequence alignment. However, multiple alignment fails to provide accurate results when a sequence comes from the twilight zone, that is, it is characterized by low (<30%) homology. To this end, we propose a novel method for prediction of secondary structure content through comprehensive sequence representation, called PSSC-core. The method uses a multiple linear regression model and introduces a comprehensive feature-based sequence representation to predict amount of helices and strands for sequences from the twilight zone. The PSSC-core method was tested and compared with two other state-of-the-art prediction methods on a set of 2187 twilight zone sequences. The results indicate that our method provides better predictions for both helix and strand content. The PSSC-core is shown to provide statistically significantly better results when compared with the competing methods, reducing the prediction error by 5-7% for helix and 7-9% for strand content predictions. The proposed feature-based sequence representation uses a comprehensive set of physicochemical properties that are custom-designed for each of the helix and strand content predictions. It includes composition and composition moment vectors, frequency of tetra-peptides associated with helical and strand conformations, various property-based groups like exchange groups, chemical groups of the side chains and hydrophobic group, auto-correlations based on hydrophobicity, side-chain masses, hydropathy, and conformational patterns for beta-sheets. The PSSC-core method provides an alternative for predicting the secondary structure content that can be used to validate and constrain results of other structure prediction methods. At the same time, it also provides useful insight into design of successful protein sequence representations that can be used in developing new methods related to prediction of different aspects of the secondary protein structure. (c) 2007 Wiley-Liss, Inc.

De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.

PubMed

Liu, Xiaowen; Dekker, Lennard J M; Wu, Si; Vanduijn, Martijn M; Luider, Theo M; Tolić, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Paša-Tolić, Ljiljana; Pevzner, Pavel A

2014-07-03

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

Determinants of the rate of protein sequence evolution

PubMed Central

Zhang, Jianzhi; Yang, Jian-Rong

2015-01-01

The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what constitutes functional constraint has remained unclear. The increasing availability of genomic data has allowed for much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses have identified multiple mechanisms behind these observations and demonstrated a prominent role that selection against errors in molecular and cellular processes plays in protein evolution. PMID:26055156

An intuitive graphical webserver for multiple-choice protein sequence search.

PubMed

Banky, Daniel; Szalkai, Balazs; Grolmusz, Vince

2014-04-10

Every day tens of thousands of sequence searches and sequence alignment queries are submitted to webservers. The capitalized word "BLAST" becomes a verb, describing the act of performing sequence search and alignment. However, if one needs to search for sequences that contain, for example, two hydrophobic and three polar residues at five given positions, the query formation on the most frequently used webservers will be difficult. Some servers support the formation of queries with regular expressions, but most of the users are unfamiliar with their syntax. Here we present an intuitive, easily applicable webserver, the Protein Sequence Analysis server, that allows the formation of multiple choice queries by simply drawing the residues to their positions; if more than one residue are drawn to the same position, then they will be nicely stacked on the user interface, indicating the multiple choice at the given position. This computer-game-like interface is natural and intuitive, and the coloring of the residues makes possible to form queries requiring not just certain amino acids in the given positions, but also small nonpolar, negatively charged, hydrophobic, positively charged, or polar ones. The webserver is available at http://psa.pitgroup.org. Copyright © 2014 Elsevier B.V. All rights reserved.

Conservation of coevolving protein interfaces bridges prokaryote-eukaryote homologies in the twilight zone.

PubMed

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-12-27

Protein-protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein-protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein-protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein-protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach.

Deep sequencing methods for protein engineering and design.

PubMed

Wrenbeck, Emily E; Faber, Matthew S; Whitehead, Timothy A

2017-08-01

The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances. Copyright © 2016 Elsevier Ltd. All rights reserved.

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

PubMed Central

2012-01-01

Background Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families. Results The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function. Conclusions Our results demonstrate that the method we present here using a k-modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family. PMID:22793672

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring.

PubMed

Durston, Kirk K; Chiu, David Ky; Wong, Andrew Kc; Li, Gary Cl

2012-07-13

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families. The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function. Our results demonstrate that the method we present here using a k-modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

PubMed Central

1994-01-01

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide. PMID:8034740

Design of Protein Multi-specificity Using an Independent Sequence Search Reduces the Barrier to Low Energy Sequences

PubMed Central

Sevy, Alexander M.; Jacobs, Tim M.; Crowe, James E.; Meiler, Jens

2015-01-01

Computational protein design has found great success in engineering proteins for thermodynamic stability, binding specificity, or enzymatic activity in a ‘single state’ design (SSD) paradigm. Multi-specificity design (MSD), on the other hand, involves considering the stability of multiple protein states simultaneously. We have developed a novel MSD algorithm, which we refer to as REstrained CONvergence in multi-specificity design (RECON). The algorithm allows each state to adopt its own sequence throughout the design process rather than enforcing a single sequence on all states. Convergence to a single sequence is encouraged through an incrementally increasing convergence restraint for corresponding positions. Compared to MSD algorithms that enforce (constrain) an identical sequence on all states the energy landscape is simplified, which accelerates the search drastically. As a result, RECON can readily be used in simulations with a flexible protein backbone. We have benchmarked RECON on two design tasks. First, we designed antibodies derived from a common germline gene against their diverse targets to assess recovery of the germline, polyspecific sequence. Second, we design “promiscuous”, polyspecific proteins against all binding partners and measure recovery of the native sequence. We show that RECON is able to efficiently recover native-like, biologically relevant sequences in this diverse set of protein complexes. PMID:26147100

Metagenome assembly through clustering of next-generation sequencing data using protein sequences.

PubMed

Sim, Mikang; Kim, Jaebum

2015-02-01

The study of environmental microbial communities, called metagenomics, has gained a lot of attention because of the recent advances in next-generation sequencing (NGS) technologies. Microbes play a critical role in changing their environments, and the mode of their effect can be solved by investigating metagenomes. However, the difficulty of metagenomes, such as the combination of multiple microbes and different species abundance, makes metagenome assembly tasks more challenging. In this paper, we developed a new metagenome assembly method by utilizing protein sequences, in addition to the NGS read sequences. Our method (i) builds read clusters by using mapping information against available protein sequences, and (ii) creates contig sequences by finding consensus sequences through probabilistic choices from the read clusters. By using simulated NGS read sequences from real microbial genome sequences, we evaluated our method in comparison with four existing assembly programs. We found that our method could generate relatively long and accurate metagenome assemblies, indicating that the idea of using protein sequences, as a guide for the assembly, is promising. Copyright © 2015 Elsevier B.V. All rights reserved.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

Evolutionary profiles from the QR factorization of multiple sequence alignments

PubMed Central

Sethi, Anurag; O'Donoghue, Patrick; Luthey-Schulten, Zaida

2005-01-01

We present an algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of the homologous group. The method, based on the multidimensional QR factorization of numerically encoded multiple sequence alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. We observe a general trend that these smaller, more evolutionarily balanced profiles have comparable and, in many cases, better performance in database searches than conventional profiles containing hundreds of sequences, constructed in an iterative and computationally intensive procedure. For more diverse families or superfamilies, with sequence identity <30%, structural alignments, based purely on the geometry of the protein structures, provide better alignments than pure sequence-based methods. Merging the structure and sequence information allows the construction of accurate profiles for distantly related groups. These structure-based profiles outperformed other sequence-based methods for finding distant homologs and were used to identify a putative class II cysteinyl-tRNA synthetase (CysRS) in several archaea that eluded previous annotation studies. Phylogenetic analysis showed the putative class II CysRSs to be a monophyletic group and homology modeling revealed a constellation of active site residues similar to that in the known class I CysRS. PMID:15741270

MoonProt: a database for proteins that are known to moonlight

PubMed Central

Mani, Mathew; Chen, Chang; Amblee, Vaishak; Liu, Haipeng; Mathur, Tanu; Zwicke, Grant; Zabad, Shadi; Patel, Bansi; Thakkar, Jagravi; Jeffery, Constance J.

2015-01-01

Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple biochemical functions that are not due to gene fusions, multiple RNA splice variants or pleiotropic effects. The known moonlighting proteins perform a variety of diverse functions in many different cell types and species, and information about their structures and functions is scattered in many publications. We have constructed the manually curated, searchable, internet-based MoonProt Database (http://www.moonlightingproteins.org) with information about the over 200 proteins that have been experimentally verified to be moonlighting proteins. The availability of this organized information provides a more complete picture of what is currently known about moonlighting proteins. The database will also aid researchers in other fields, including determining the functions of genes identified in genome sequencing projects, interpreting data from proteomics projects and annotating protein sequence and structural databases. In addition, information about the structures and functions of moonlighting proteins can be helpful in understanding how novel protein functional sites evolved on an ancient protein scaffold, which can also help in the design of proteins with novel functions. PMID:25324305

Protein Sectors: Statistical Coupling Analysis versus Conservation

PubMed Central

Teşileanu, Tiberiu; Colwell, Lucy J.; Leibler, Stanislas

2015-01-01

Statistical coupling analysis (SCA) is a method for analyzing multiple sequence alignments that was used to identify groups of coevolving residues termed “sectors”. The method applies spectral analysis to a matrix obtained by combining correlation information with sequence conservation. It has been asserted that the protein sectors identified by SCA are functionally significant, with different sectors controlling different biochemical properties of the protein. Here we reconsider the available experimental data and note that it involves almost exclusively proteins with a single sector. We show that in this case sequence conservation is the dominating factor in SCA, and can alone be used to make statistically equivalent functional predictions. Therefore, we suggest shifting the experimental focus to proteins for which SCA identifies several sectors. Correlations in protein alignments, which have been shown to be informative in a number of independent studies, would then be less dominated by sequence conservation. PMID:25723535

Phylogenetic Characterization of Transport Protein Superfamilies: Superiority of SuperfamilyTree Programs over Those Based on Multiple Alignments

PubMed Central

Chen, Jonathan S.; Reddy, Vamsee; Chen, Joshua H.; Shlykov, Maksim A.; Zheng, Wei Hao; Cho, Jaehoon; Yen, Ming Ren; Saier, Milton H.

2012-01-01

Transport proteins function in the translocation of ions, solutes and macromolecules across cellular and organellar membranes. These integral membrane proteins fall into >600 families as tabulated in the Transporter Classification Database (www.tcdb.org). Recent studies, some of which are reported here, define distant phylogenetic relationships between families with the creation of superfamilies. Several of these are analyzed using a novel set of programs designed to allow reliable prediction of phylogenetic trees when sequence divergence is too great to allow the use of multiple alignments. These new programs, called SuperfamilyTree1 and 2 (SFT1 and 2), allow display of protein and family relationships, respectively, based on thousands of comparative BLAST scores rather than multiple alignments. Superfamilies analyzed include: (1) Aerolysins, (2) RTX Toxins, (3) Defensins, (4) Ion Transporters, (5) Bile/Arsenite/Riboflavin Transporters, (6) Cation: Proton Antiporters, and (7) the Glucose/Fructose/Lactose superfamily within the prokaryotic phosphoenol pyruvate-dependent Phosphotransferase System. In addition to defining the phylogenetic relationships of the proteins and families within these seven superfamilies, evidence is provided showing that the SFT programs outperform programs that are based on multiple alignments whenever sequence divergence of superfamily members is extensive. The SFT programs should be applicable to virtually any superfamily of proteins or nucleic acids. PMID:22286036

Pfarao: a web application for protein family analysis customized for cytoskeletal and motor proteins (CyMoBase).

PubMed

Odronitz, Florian; Kollmar, Martin

2006-11-29

Annotation of protein sequences of eukaryotic organisms is crucial for the understanding of their function in the cell. Manual annotation is still by far the most accurate way to correctly predict genes. The classification of protein sequences, their phylogenetic relation and the assignment of function involves information from various sources. This often leads to a collection of heterogeneous data, which is hard to track. Cytoskeletal and motor proteins consist of large and diverse superfamilies comprising up to several dozen members per organism. Up to date there is no integrated tool available to assist in the manual large-scale comparative genomic analysis of protein families. Pfarao (Protein Family Application for Retrieval, Analysis and Organisation) is a database driven online working environment for the analysis of manually annotated protein sequences and their relationship. Currently, the system can store and interrelate a wide range of information about protein sequences, species, phylogenetic relations and sequencing projects as well as links to literature and domain predictions. Sequences can be imported from multiple sequence alignments that are generated during the annotation process. A web interface allows to conveniently browse the database and to compile tabular and graphical summaries of its content. We implemented a protein sequence-centric web application to store, organize, interrelate, and present heterogeneous data that is generated in manual genome annotation and comparative genomics. The application has been developed for the analysis of cytoskeletal and motor proteins (CyMoBase) but can easily be adapted for any protein.

Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

PubMed Central

Du, Pufeng; Wang, Lusheng

2014-01-01

One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods. PMID:24466278

Multiple DNA and protein sequence alignment on a workstation and a supercomputer.

PubMed

Tajima, K

1988-11-01

This paper describes a multiple alignment method using a workstation and supercomputer. The method is based on the alignment of a set of aligned sequences with the new sequence, and uses a recursive procedure of such alignment. The alignment is executed in a reasonable computation time on diverse levels from a workstation to a supercomputer, from the viewpoint of alignment results and computational speed by parallel processing. The application of the algorithm is illustrated by several examples of multiple alignment of 12 amino acid and DNA sequences of HIV (human immunodeficiency virus) env genes. Colour graphic programs on a workstation and parallel processing on a supercomputer are discussed.

Identifying functionally informative evolutionary sequence profiles.

PubMed

Gil, Nelson; Fiser, Andras

2018-04-15

Multiple sequence alignments (MSAs) can provide essential input to many bioinformatics applications, including protein structure prediction and functional annotation. However, the optimal selection of sequences to obtain biologically informative MSAs for such purposes is poorly explored, and has traditionally been performed manually. We present Selection of Alignment by Maximal Mutual Information (SAMMI), an automated, sequence-based approach to objectively select an optimal MSA from a large set of alternatives sampled from a general sequence database search. The hypothesis of this approach is that the mutual information among MSA columns will be maximal for those MSAs that contain the most diverse set possible of the most structurally and functionally homogeneous protein sequences. SAMMI was tested to select MSAs for functional site residue prediction by analysis of conservation patterns on a set of 435 proteins obtained from protein-ligand (peptides, nucleic acids and small substrates) and protein-protein interaction databases. Availability and implementation: A freely accessible program, including source code, implementing SAMMI is available at https://github.com/nelsongil92/SAMMI.git. andras.fiser@einstein.yu.edu. Supplementary data are available at Bioinformatics online.

MOCASSIN-prot: A multi-objective clustering approach for protein similarity networks

USDA-ARS?s Scientific Manuscript database

Motivation: Proteins often include multiple conserved domains. Various evolutionary events including duplication and loss of domains, domain shuffling, as well as sequence divergence contribute to generating complexities in protein structures, and consequently, in their functions. The evolutionary h...

Protein fold recognition using geometric kernel data fusion.

PubMed

Zakeri, Pooya; Jeuris, Ben; Vandebril, Raf; Moreau, Yves

2014-07-01

Various approaches based on features extracted from protein sequences and often machine learning methods have been used in the prediction of protein folds. Finding an efficient technique for integrating these different protein features has received increasing attention. In particular, kernel methods are an interesting class of techniques for integrating heterogeneous data. Various methods have been proposed to fuse multiple kernels. Most techniques for multiple kernel learning focus on learning a convex linear combination of base kernels. In addition to the limitation of linear combinations, working with such approaches could cause a loss of potentially useful information. We design several techniques to combine kernel matrices by taking more involved, geometry inspired means of these matrices instead of convex linear combinations. We consider various sequence-based protein features including information extracted directly from position-specific scoring matrices and local sequence alignment. We evaluate our methods for classification on the SCOP PDB-40D benchmark dataset for protein fold recognition. The best overall accuracy on the protein fold recognition test set obtained by our methods is ∼ 86.7%. This is an improvement over the results of the best existing approach. Moreover, our computational model has been developed by incorporating the functional domain composition of proteins through a hybridization model. It is observed that by using our proposed hybridization model, the protein fold recognition accuracy is further improved to 89.30%. Furthermore, we investigate the performance of our approach on the protein remote homology detection problem by fusing multiple string kernels. The MATLAB code used for our proposed geometric kernel fusion frameworks are publicly available at http://people.cs.kuleuven.be/∼raf.vandebril/homepage/software/geomean.php?menu=5/. © The Author 2014. Published by Oxford University Press.

Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein

NASA Astrophysics Data System (ADS)

Guinn, Emily J.; Jagannathan, Bharat; Marqusee, Susan

2015-04-01

A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.

CoSMoS: Conserved Sequence Motif Search in the proteome

PubMed Central

Liu, Xiao I; Korde, Neeraj; Jakob, Ursula; Leichert, Lars I

2006-01-01

Background With the ever-increasing number of gene sequences in the public databases, generating and analyzing multiple sequence alignments becomes increasingly time consuming. Nevertheless it is a task performed on a regular basis by researchers in many labs. Results We have now created a database called CoSMoS to find the occurrences and at the same time evaluate the significance of sequence motifs and amino acids encoded in the whole genome of the model organism Escherichia coli K12. We provide a precomputed set of multiple sequence alignments for each individual E. coli protein with all of its homologues in the RefSeq database. The alignments themselves, information about the occurrence of sequence motifs together with information on the conservation of each of the more than 1.3 million amino acids encoded in the E. coli genome can be accessed via the web interface of CoSMoS. Conclusion CoSMoS is a valuable tool to identify highly conserved sequence motifs, to find regions suitable for mutational studies in functional analyses and to predict important structural features in E. coli proteins. PMID:16433915

BEAUTY: an enhanced BLAST-based search tool that integrates multiple biological information resources into sequence similarity search results.

PubMed

Worley, K C; Wiese, B A; Smith, R F

1995-09-01

BEAUTY (BLAST enhanced alignment utility) is an enhanced version of the NCBI's BLAST data base search tool that facilitates identification of the functions of matched sequences. We have created new data bases of conserved regions and functional domains for protein sequences in NCBI's Entrez data base, and BEAUTY allows this information to be incorporated directly into BLAST search results. A Conserved Regions Data Base, containing the locations of conserved regions within Entrez protein sequences, was constructed by (1) clustering the entire data base into families, (2) aligning each family using our PIMA multiple sequence alignment program, and (3) scanning the multiple alignments to locate the conserved regions within each aligned sequence. A separate Annotated Domains Data Base was constructed by extracting the locations of all annotated domains and sites from sequences represented in the Entrez, PROSITE, BLOCKS, and PRINTS data bases. BEAUTY performs a BLAST search of those Entrez sequences with conserved regions and/or annotated domains. BEAUTY then uses the information from the Conserved Regions and Annotated Domains data bases to generate, for each matched sequence, a schematic display that allows one to directly compare the relative locations of (1) the conserved regions, (2) annotated domains and sites, and (3) the locally aligned regions matched in the BLAST search. In addition, BEAUTY search results include World-Wide Web hypertext links to a number of external data bases that provide a variety of additional types of information on the function of matched sequences. This convenient integration of protein families, conserved regions, annotated domains, alignment displays, and World-Wide Web resources greatly enhances the biological informativeness of sequence similarity searches. BEAUTY searches can be performed remotely on our system using the "BCM Search Launcher" World-Wide Web pages (URL is < http:/ /gc.bcm.tmc.edu:8088/ search-launcher/launcher.html > ).

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis).

PubMed

Darville, Lancia N F; Merchant, Mark E; Maccha, Venkata; Siddavarapu, Vivekananda Reddy; Hasan, Azeem; Murray, Kermit K

2012-02-01

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates. Copyright © 2011 Elsevier Inc. All rights reserved.

Defining and predicting structurally conserved regions in protein superfamilies

PubMed Central

Huang, Ivan K.; Grishin, Nick V.

2013-01-01

Motivation: The structures of homologous proteins are generally better conserved than their sequences. This phenomenon is demonstrated by the prevalence of structurally conserved regions (SCRs) even in highly divergent protein families. Defining SCRs requires the comparison of two or more homologous structures and is affected by their availability and divergence, and our ability to deduce structurally equivalent positions among them. In the absence of multiple homologous structures, it is necessary to predict SCRs of a protein using information from only a set of homologous sequences and (if available) a single structure. Accurate SCR predictions can benefit homology modelling and sequence alignment. Results: Using pairwise DaliLite alignments among a set of homologous structures, we devised a simple measure of structural conservation, termed structural conservation index (SCI). SCI was used to distinguish SCRs from non-SCRs. A database of SCRs was compiled from 386 SCOP superfamilies containing 6489 protein domains. Artificial neural networks were then trained to predict SCRs with various features deduced from a single structure and homologous sequences. Assessment of the predictions via a 5-fold cross-validation method revealed that predictions based on features derived from a single structure perform similarly to ones based on homologous sequences, while combining sequence and structural features was optimal in terms of accuracy (0.755) and Matthews correlation coefficient (0.476). These results suggest that even without information from multiple structures, it is still possible to effectively predict SCRs for a protein. Finally, inspection of the structures with the worst predictions pinpoints difficulties in SCR definitions. Availability: The SCR database and the prediction server can be found at http://prodata.swmed.edu/SCR. Contact: 91huangi@gmail.com or grishin@chop.swmed.edu Supplementary information: Supplementary data are available at Bioinformatics Online PMID:23193223

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information.

PubMed

Song, Jiangning; Burrage, Kevin; Yuan, Zheng; Huber, Thomas

2006-03-09

The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

PubMed

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes).

PubMed

Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-09-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

PubMed Central

Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-01-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341

Automatic prediction of protein domains from sequence information using a hybrid learning system.

PubMed

Nagarajan, Niranjan; Yona, Golan

2004-06-12

We describe a novel method for detecting the domain structure of a protein from sequence information alone. The method is based on analyzing multiple sequence alignments that are derived from a database search. Multiple measures are defined to quantify the domain information content of each position along the sequence and are combined into a single predictor using a neural network. The output is further smoothed and post-processed using a probabilistic model to predict the most likely transition positions between domains. The method was assessed using the domain definitions in SCOP and CATH for proteins of known structure and was compared with several other existing methods. Our method performs well both in terms of accuracy and sensitivity. It improves significantly over the best methods available, even some of the semi-manual ones, while being fully automatic. Our method can also be used to suggest and verify domain partitions based on structural data. A few examples of predicted domain definitions and alternative partitions, as suggested by our method, are also discussed. An online domain-prediction server is available at http://biozon.org/tools/domains/

Phenotype–genotype correlation in Hirschsprung disease is illuminated by comparative analysis of the RET protein sequence

PubMed Central

Kashuk, Carl S.; Stone, Eric A.; Grice, Elizabeth A.; Portnoy, Matthew E.; Green, Eric D.; Sidow, Arend; Chakravarti, Aravinda; McCallion, Andrew S.

2005-01-01

The ability to discriminate between deleterious and neutral amino acid substitutions in the genes of patients remains a significant challenge in human genetics. The increasing availability of genomic sequence data from multiple vertebrate species allows inclusion of sequence conservation and physicochemical properties of residues to be used for functional prediction. In this study, the RET receptor tyrosine kinase serves as a model disease gene in which a broad spectrum (≥116) of disease-associated mutations has been identified among patients with Hirschsprung disease and multiple endocrine neoplasia type 2. We report the alignment of the human RET protein sequence with the orthologous sequences of 12 non-human vertebrates (eight mammalian, one avian, and three teleost species), their comparative analysis, the evolutionary topology of the RET protein, and predicted tolerance for all published missense mutations. We show that, although evolutionary conservation alone provides significant information to predict the effect of a RET mutation, a model that combines comparative sequence data with analysis of physiochemical properties in a quantitative framework provides far greater accuracy. Although the ability to discern the impact of a mutation is imperfect, our analyses permit substantial discrimination between predicted functional classes of RET mutations and disease severity even for a multigenic disease such as Hirschsprung disease. PMID:15956201

Conservation of coevolving protein interfaces bridges prokaryote–eukaryote homologies in the twilight zone

PubMed Central

Rodriguez-Rivas, Juan; Marsili, Simone; Juan, David; Valencia, Alfonso

2016-01-01

Protein–protein interactions are fundamental for the proper functioning of the cell. As a result, protein interaction surfaces are subject to strong evolutionary constraints. Recent developments have shown that residue coevolution provides accurate predictions of heterodimeric protein interfaces from sequence information. So far these approaches have been limited to the analysis of families of prokaryotic complexes for which large multiple sequence alignments of homologous sequences can be compiled. We explore the hypothesis that coevolution points to structurally conserved contacts at protein–protein interfaces, which can be reliably projected to homologous complexes with distantly related sequences. We introduce a domain-centered protocol to study the interplay between residue coevolution and structural conservation of protein–protein interfaces. We show that sequence-based coevolutionary analysis systematically identifies residue contacts at prokaryotic interfaces that are structurally conserved at the interface of their eukaryotic counterparts. In turn, this allows the prediction of conserved contacts at eukaryotic protein–protein interfaces with high confidence using solely mutational patterns extracted from prokaryotic genomes. Even in the context of high divergence in sequence (the twilight zone), where standard homology modeling of protein complexes is unreliable, our approach provides sequence-based accurate information about specific details of protein interactions at the residue level. Selected examples of the application of prokaryotic coevolutionary analysis to the prediction of eukaryotic interfaces further illustrate the potential of this approach. PMID:27965389

Pfarao: a web application for protein family analysis customized for cytoskeletal and motor proteins (CyMoBase)

PubMed Central

Odronitz, Florian; Kollmar, Martin

2006-01-01

Background Annotation of protein sequences of eukaryotic organisms is crucial for the understanding of their function in the cell. Manual annotation is still by far the most accurate way to correctly predict genes. The classification of protein sequences, their phylogenetic relation and the assignment of function involves information from various sources. This often leads to a collection of heterogeneous data, which is hard to track. Cytoskeletal and motor proteins consist of large and diverse superfamilies comprising up to several dozen members per organism. Up to date there is no integrated tool available to assist in the manual large-scale comparative genomic analysis of protein families. Description Pfarao (Protein Family Application for Retrieval, Analysis and Organisation) is a database driven online working environment for the analysis of manually annotated protein sequences and their relationship. Currently, the system can store and interrelate a wide range of information about protein sequences, species, phylogenetic relations and sequencing projects as well as links to literature and domain predictions. Sequences can be imported from multiple sequence alignments that are generated during the annotation process. A web interface allows to conveniently browse the database and to compile tabular and graphical summaries of its content. Conclusion We implemented a protein sequence-centric web application to store, organize, interrelate, and present heterogeneous data that is generated in manual genome annotation and comparative genomics. The application has been developed for the analysis of cytoskeletal and motor proteins (CyMoBase) but can easily be adapted for any protein. PMID:17134497

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins

PubMed Central

Vanhoutteghem, Amandine; Maciejewski-Duval, Anna; Bouche, Cyril; Delhomme, Brigitte; Hervé, Françoise; Daubigney, Fabrice; Soubigou, Guillaume; Araki, Masatake; Araki, Kimi; Yamamura, Ken-ichi; Djian, Philippe

2009-01-01

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2. PMID:19706529

Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

PubMed Central

2012-01-01

Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain annotation by identifying those pattern residues that most distinguish each protein domain subgroup from other related subgroups. PMID:22726767

On the Role of Aggregation Prone Regions in Protein Evolution, Stability, and Enzymatic Catalysis: Insights from Diverse Analyses

PubMed Central

Buck, Patrick M.; Kumar, Sandeep; Singh, Satish K.

2013-01-01

The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity. PMID:24146608

Scop3D: three-dimensional visualization of sequence conservation.

PubMed

Vermeire, Tessa; Vermaere, Stijn; Schepens, Bert; Saelens, Xavier; Van Gucht, Steven; Martens, Lennart; Vandermarliere, Elien

2015-04-01

The integration of a protein's structure with its known sequence variation provides insight on how that protein evolves, for instance in terms of (changing) function or immunogenicity. Yet, collating the corresponding sequence variants into a multiple sequence alignment, calculating each position's conservation, and mapping this information back onto a relevant structure is not straightforward. We therefore built the Sequence Conservation on Protein 3D structure (scop3D) tool to perform these tasks automatically. The output consists of two modified PDB files in which the B-values for each position are replaced by the percentage sequence conservation, or the information entropy for each position, respectively. Furthermore, text files with absolute and relative amino acid occurrences for each position are also provided, along with snapshots of the protein from six distinct directions in space. The visualization provided by scop3D can for instance be used as an aid in vaccine development or to identify antigenic hotspots, which we here demonstrate based on an analysis of the fusion proteins of human respiratory syncytial virus and mumps virus. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evol and ProDy for bridging protein sequence evolution and structural dynamics

PubMed Central

Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R.; Bahar, Ivet

2014-01-01

Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolution profiles extracted from multiple sequence alignments of protein families with their inferred dynamics. Availability and implementation: ProDy and Evol are open-source and freely available under MIT License from http://prody.csb.pitt.edu/. Contact: bahar@pitt.edu PMID:24849577

"Multiple partial recognitions in dynamic equilibrium" in the binding sites of proteins form the molecular basis of promiscuous recognition of structurally diverse ligands.

PubMed

Kohda, Daisuke

2018-04-01

Promiscuous recognition of ligands by proteins is as important as strict recognition in numerous biological processes. In living cells, many short, linear amino acid motifs function as targeting signals in proteins to specify the final destination of the protein transport. In general, the target signal is defined by a consensus sequence containing wild-characters, and hence represented by diverse amino acid sequences. The classical lock-and-key or induced-fit/conformational selection mechanism may not cover all aspects of the promiscuous recognition. On the basis of our crystallographic and NMR studies on the mitochondrial Tom20 protein-presequence interaction, we proposed a new hypothetical mechanism based on "a rapid equilibrium of multiple states with partial recognitions". This dynamic, multiple recognition mode enables the Tom20 receptor to recognize diverse mitochondrial presequences with nearly equal affinities. The plant Tom20 is evolutionally unrelated to the animal Tom20 in our study, but is a functional homolog of the animal/fungal Tom20. NMR studies by another research group revealed that the presequence binding by the plant Tom20 was not fully explained by simple interaction modes, suggesting the presence of a similar dynamic, multiple recognition mode. Circumstantial evidence also suggested that similar dynamic mechanisms may be applicable to other promiscuous recognitions of signal peptides by the SRP54/Ffh and SecA proteins.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

PubMed

Shahinyan, Grigor; Margaryan, Armine; Panosyan, Hovik; Trchounian, Armen

2017-05-02

Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

PubMed Central

Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

1989-01-01

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501

Protein Annotators' Assistant: A Novel Application of Information Retrieval Techniques.

ERIC Educational Resources Information Center

Wise, Michael J.

2000-01-01

Protein Annotators' Assistant (PAA) is a software system which assists protein annotators in assigning functions to newly sequenced proteins. PAA employs a number of information retrieval techniques in a novel setting and is thus related to text categorization, where multiple categories may be suggested, except that in this case none of the…

Biomolecular characterization and protein sequences of the Campanian hadrosaur B. canadensis.

PubMed

Schweitzer, Mary H; Zheng, Wenxia; Organ, Chris L; Avci, Recep; Suo, Zhiyong; Freimark, Lisa M; Lebleu, Valerie S; Duncan, Michael B; Vander Heiden, Matthew G; Neveu, John M; Lane, William S; Cottrell, John S; Horner, John R; Cantley, Lewis C; Kalluri, Raghu; Asara, John M

2009-05-01

Molecular preservation in non-avian dinosaurs is controversial. We present multiple lines of evidence that endogenous proteinaceous material is preserved in bone fragments and soft tissues from an 80-million-year-old Campanian hadrosaur, Brachylophosaurus canadensis [Museum of the Rockies (MOR) 2598]. Microstructural and immunological data are consistent with preservation of multiple bone matrix and vessel proteins, and phylogenetic analyses of Brachylophosaurus collagen sequenced by mass spectrometry robustly support the bird-dinosaur clade, consistent with an endogenous source for these collagen peptides. These data complement earlier results from Tyrannosaurus rex (MOR 1125) and confirm that molecular preservation in Cretaceous dinosaurs is not a unique event.

ProtDec-LTR2.0: an improved method for protein remote homology detection by combining pseudo protein and supervised Learning to Rank.

PubMed

Chen, Junjie; Guo, Mingyue; Li, Shumin; Liu, Bin

2017-11-01

As one of the most important tasks in protein sequence analysis, protein remote homology detection is critical for both basic research and practical applications. Here, we present an effective web server for protein remote homology detection called ProtDec-LTR2.0 by combining ProtDec-Learning to Rank (LTR) and pseudo protein representation. Experimental results showed that the detection performance is obviously improved. The web server provides a user-friendly interface to explore the sequence and structure information of candidate proteins and find their conserved domains by launching a multiple sequence alignment tool. The web server is free and open to all users with no login requirement at http://bioinformatics.hitsz.edu.cn/ProtDec-LTR2.0/. bliu@hit.edu.cn. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Simultaneous phylogeny reconstruction and multiple sequence alignment

PubMed Central

Yue, Feng; Shi, Jian; Tang, Jijun

2009-01-01

Background A phylogeny is the evolutionary history of a group of organisms. To date, sequence data is still the most used data type for phylogenetic reconstruction. Before any sequences can be used for phylogeny reconstruction, they must be aligned, and the quality of the multiple sequence alignment has been shown to affect the quality of the inferred phylogeny. At the same time, all the current multiple sequence alignment programs use a guide tree to produce the alignment and experiments showed that good guide trees can significantly improve the multiple alignment quality. Results We devise a new algorithm to simultaneously align multiple sequences and search for the phylogenetic tree that leads to the best alignment. We also implemented the algorithm as a C program package, which can handle both DNA and protein data and can take simple cost model as well as complex substitution matrices, such as PAM250 or BLOSUM62. The performance of the new method are compared with those from other popular multiple sequence alignment tools, including the widely used programs such as ClustalW and T-Coffee. Experimental results suggest that this method has good performance in terms of both phylogeny accuracy and alignment quality. Conclusion We present an algorithm to align multiple sequences and reconstruct the phylogenies that minimize the alignment score, which is based on an efficient algorithm to solve the median problems for three sequences. Our extensive experiments suggest that this method is very promising and can produce high quality phylogenies and alignments. PMID:19208110

Isolation and characterization of adrenoleukodystrophy protein (ALDP) related sequences in the human genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geraghty, M.T.; Stetten, G.; Kearns, W.

1994-09-01

X-linked adrenoleukodystrophy (ALD) is a disorder of peroxisomal {beta}-oxidation of very long chain fatty acids. It presents either as progressive dementia in childhood or as progressive paraparesis in later years. Adrenal insufficiency occurs in both phenotypes. The gene of the ALD protein has been mapped to Xq28 and has recently been cloned and characterized. The ALD protein has significant homology to the peroxisomal membrane protein, PMP70 and belongs to the ATP binding cassette superfamily of transporters. We screened a human genomic library with an ALDP cDNA and isolated 5 different but highly similar clones containing sequences corresponding to the 3{prime}more » end of the ALDP gene. Comparison of the sequences over the region corresponding to exon 9 through the 3{prime} end of the ALDP gene reveals {approximately}96% nucleotide identity in both exonic and intronic regions. Splice sites and open reading frames are maintained. Using both FISH and human-rodent DNA mapping panels, we positively assign these ALDP-related sequences to chromosomes 2, 16 and 22, and provisionally to 1 and 20. Southern blot of primate DNA probed with a partial ALDP cDNA (exon 2-10) shows that expansion of ALDP-related sequences occurred in higher primates (chimp, gorilla and human). Although Northern blots show multiple ALDP-hybridizing transcripts in certain tissues, we have no evidence to date for expression of these ALDP-related sequences. In conclusion, our data show there has been an unusual and recent dispersal to multiple chromosomes of structural gene sequences related to the ALDP gene. The functional significance of these sequences remains to be determined but their existence complicates PCR and mutation analysis of the ALDP gene.« less

Conservation of hot regions in protein-protein interaction in evolution.

PubMed

Hu, Jing; Li, Jiarui; Chen, Nansheng; Zhang, Xiaolong

2016-11-01

The hot regions of protein-protein interactions refer to the active area which formed by those most important residues to protein combination process. With the research development on protein interactions, lots of predicted hot regions can be discovered efficiently by intelligent computing methods, while performing biology experiments to verify each every prediction is hardly to be done due to the time-cost and the complexity of the experiment. This study based on the research of hot spot residue conservations, the proposed method is used to verify authenticity of predicted hot regions that using machine learning algorithm combined with protein's biological features and sequence conservation, though multiple sequence alignment, module substitute matrix and sequence similarity to create conservation scoring algorithm, and then using threshold module to verify the conservation tendency of hot regions in evolution. This research work gives an effective method to verify predicted hot regions in protein-protein interactions, which also provides a useful way to deeply investigate the functional activities of protein hot regions. Copyright © 2016. Published by Elsevier Inc.

Quantiprot - a Python package for quantitative analysis of protein sequences.

PubMed

Konopka, Bogumił M; Marciniak, Marta; Dyrka, Witold

2017-07-17

The field of protein sequence analysis is dominated by tools rooted in substitution matrices and alignments. A complementary approach is provided by methods of quantitative characterization. A major advantage of the approach is that quantitative properties defines a multidimensional solution space, where sequences can be related to each other and differences can be meaningfully interpreted. Quantiprot is a software package in Python, which provides a simple and consistent interface to multiple methods for quantitative characterization of protein sequences. The package can be used to calculate dozens of characteristics directly from sequences or using physico-chemical properties of amino acids. Besides basic measures, Quantiprot performs quantitative analysis of recurrence and determinism in the sequence, calculates distribution of n-grams and computes the Zipf's law coefficient. We propose three main fields of application of the Quantiprot package. First, quantitative characteristics can be used in alignment-free similarity searches, and in clustering of large and/or divergent sequence sets. Second, a feature space defined by quantitative properties can be used in comparative studies of protein families and organisms. Third, the feature space can be used for evaluating generative models, where large number of sequences generated by the model can be compared to actually observed sequences.

The SUPERFAMILY database in 2004: additions and improvements.

PubMed

Madera, Martin; Vogel, Christine; Kummerfeld, Sarah K; Chothia, Cyrus; Gough, Julian

2004-01-01

The SUPERFAMILY database provides structural assignments to protein sequences and a framework for analysis of the results. At the core of the database is a library of profile Hidden Markov Models that represent all proteins of known structure. The library is based on the SCOP classification of proteins: each model corresponds to a SCOP domain and aims to represent an entire superfamily. We have applied the library to predicted proteins from all completely sequenced genomes (currently 154), the Swiss-Prot and TrEMBL databases and other sequence collections. Close to 60% of all proteins have at least one match, and one half of all residues are covered by assignments. All models and full results are available for download and online browsing at http://supfam.org. Users can study the distribution of their superfamily of interest across all completely sequenced genomes, investigate with which other superfamilies it combines and retrieve proteins in which it occurs. Alternatively, concentrating on a particular genome as a whole, it is possible first, to find out its superfamily composition, and secondly, to compare it with that of other genomes to detect superfamilies that are over- or under-represented. In addition, the webserver provides the following standard services: sequence search; keyword search for genomes, superfamilies and sequence identifiers; and multiple alignment of genomic, PDB and custom sequences.

Detection of alternative splice variants at the proteome level in Aspergillus flavus.

PubMed

Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

2010-03-05

Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

Protein 3D Structure Computed from Evolutionary Sequence Variation

PubMed Central

Sheridan, Robert; Hopf, Thomas A.; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

2011-01-01

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing. In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy. We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues., including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7–4.8 Å Cα-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein structures, new strategies in protein and drug design, and the identification of functional genetic variants in normal and disease genomes. PMID:22163331

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

JDet: interactive calculation and visualization of function-related conservation patterns in multiple sequence alignments and structures.

PubMed

Muth, Thilo; García-Martín, Juan A; Rausell, Antonio; Juan, David; Valencia, Alfonso; Pazos, Florencio

2012-02-15

We have implemented in a single package all the features required for extracting, visualizing and manipulating fully conserved positions as well as those with a family-dependent conservation pattern in multiple sequence alignments. The program allows, among other things, to run different methods for extracting these positions, combine the results and visualize them in protein 3D structures and sequence spaces. JDet is a multiplatform application written in Java. It is freely available, including the source code, at http://csbg.cnb.csic.es/JDet. The package includes two of our recently developed programs for detecting functional positions in protein alignments (Xdet and S3Det), and support for other methods can be added as plug-ins. A help file and a guided tutorial for JDet are also available.

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valley, Cary T.; Porter, Douglas F.; Qiu, Chen

2012-06-28

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites.more » Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.« less

Hidden Markov models of biological primary sequence information.

PubMed Central

Baldi, P; Chauvin, Y; Hunkapiller, T; McClure, M A

1994-01-01

Hidden Markov model (HMM) techniques are used to model families of biological sequences. A smooth and convergent algorithm is introduced to iteratively adapt the transition and emission parameters of the models from the examples in a given family. The HMM approach is applied to three protein families: globins, immunoglobulins, and kinases. In all cases, the models derived capture the important statistical characteristics of the family and can be used for a number of tasks, including multiple alignments, motif detection, and classification. For K sequences of average length N, this approach yields an effective multiple-alignment algorithm which requires O(KN2) operations, linear in the number of sequences. PMID:8302831

ProteinInferencer: Confident protein identification and multiple experiment comparison for large scale proteomics projects.

PubMed

Zhang, Yaoyang; Xu, Tao; Shan, Bing; Hart, Jonathan; Aslanian, Aaron; Han, Xuemei; Zong, Nobel; Li, Haomin; Choi, Howard; Wang, Dong; Acharya, Lipi; Du, Lisa; Vogt, Peter K; Ping, Peipei; Yates, John R

2015-11-03

Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein-protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015. Published by Elsevier B.V.

Regulation of Bacteria-Induced Intercellular Adhesion Molecule-1 by CCAAT/Enhancer Binding Proteins

PubMed Central

Manzel, Lori J.; Chin, Cecilia L.; Behlke, Mark A.; Look, Dwight C.

2009-01-01

Direct interaction between bacteria and epithelial cells may initiate or amplify the airway response through induction of epithelial defense gene expression by nuclear factor-κB (NF-κB). However, multiple signaling pathways modify NF-κB effects to modulate gene expression. In this study, the effects of CCAAT/enhancer binding protein (C/EBP) family members on induction of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) was examined in primary cultures of human tracheobronchial epithelial cells incubated with nontypeable Haemophilus influenzae. Increased ICAM-1 gene transcription in response to H. influenzae required gene sequences located at −200 to −135 in the 5′-flanking region that contain a C/EBP-binding sequence immediately upstream of the NF-κB enhancer site. Constitutive C/EBPβ was found to have an important role in epithelial cell ICAM-1 regulation, while the adjacent NF-κB sequence binds the RelA/p65 and NF-κB1/p50 members of the NF-κB family to induce ICAM-1 expression in response to H. influenzae. The expression of C/EBP proteins is not regulated by p38 mitogen-activated protein kinase activation, but p38 affects gene transcription by increasing the binding of TATA-binding protein to TATA-box–containing gene sequences. Epithelial cell ICAM-1 expression in response to H. influenzae was decreased by expressing dominant-negative protein or RNA interference against C/EBPβ, confirming its role in ICAM-1 regulation. Although airway epithelial cells express multiple constitutive and inducible C/EBP family members that bind C/EBP sequences, the results indicate that C/EBPβ plays a central role in modulation of NF-κB–dependent defense gene expression in human airway epithelial cells after exposure to H. influenzae. PMID:18703796

eShadow: A tool for comparing closely related sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ovcharenko, Ivan; Boffelli, Dario; Loots, Gabriela G.

2004-01-15

Primate sequence comparisons are difficult to interpret due to the high degree of sequence similarity shared between such closely related species. Recently, a novel method, phylogenetic shadowing, has been pioneered for predicting functional elements in the human genome through the analysis of multiple primate sequence alignments. We have expanded this theoretical approach to create a computational tool, eShadow, for the identification of elements under selective pressure in multiple sequence alignments of closely related genomes, such as in comparisons of human to primate or mouse to rat DNA. This tool integrates two different statistical methods and allows for the dynamic visualizationmore » of the resulting conservation profile. eShadow also includes a versatile optimization module capable of training the underlying Hidden Markov Model to differentially predict functional sequences. This module grants the tool high flexibility in the analysis of multiple sequence alignments and in comparing sequences with different divergence rates. Here, we describe the eShadow comparative tool and its potential uses for analyzing both multiple nucleotide and protein alignments to predict putative functional elements. The eShadow tool is publicly available at http://eshadow.dcode.org/« less

A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities.

PubMed

Bastien, Olivier; Ortet, Philippe; Roy, Sylvaine; Maréchal, Eric

2005-03-10

Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction. We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

Low-pass sequencing for microbial comparative genomics

PubMed Central

Goo, Young Ah; Roach, Jared; Glusman, Gustavo; Baliga, Nitin S; Deutsch, Kerry; Pan, Min; Kennedy, Sean; DasSarma, Shiladitya; Victor Ng, Wailap; Hood, Leroy

2004-01-01

Background We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. Results As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. Conclusion Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics. PMID:14718067

BCM Search Launcher--an integrated interface to molecular biology data base search and analysis services available on the World Wide Web.

PubMed

Smith, R F; Wiese, B A; Wojzynski, M K; Davison, D B; Worley, K C

1996-05-01

The BCM Search Launcher is an integrated set of World Wide Web (WWW) pages that organize molecular biology-related search and analysis services available on the WWW by function, and provide a single point of entry for related searches. The Protein Sequence Search Page, for example, provides a single sequence entry form for submitting sequences to WWW servers that offer remote access to a variety of different protein sequence search tools, including BLAST, FASTA, Smith-Waterman, BEAUTY, PROSITE, and BLOCKS searches. Other Launch pages provide access to (1) nucleic acid sequence searches, (2) multiple and pair-wise sequence alignments, (3) gene feature searches, (4) protein secondary structure prediction, and (5) miscellaneous sequence utilities (e.g., six-frame translation). The BCM Search Launcher also provides a mechanism to extend the utility of other WWW services by adding supplementary hypertext links to results returned by remote servers. For example, links to the NCBI's Entrez data base and to the Sequence Retrieval System (SRS) are added to search results returned by the NCBI's WWW BLAST server. These links provide easy access to auxiliary information, such as Medline abstracts, that can be extremely helpful when analyzing BLAST data base hits. For new or infrequent users of sequence data base search tools, we have preset the default search parameters to provide the most informative first-pass sequence analysis possible. We have also developed a batch client interface for Unix and Macintosh computers that allows multiple input sequences to be searched automatically as a background task, with the results returned as individual HTML documents directly to the user's system. The BCM Search Launcher and batch client are available on the WWW at URL http:@gc.bcm.tmc.edu:8088/search-launcher.html.

Protein contact prediction by integrating deep multiple sequence alignments, coevolution and machine learning.

PubMed

Adhikari, Badri; Hou, Jie; Cheng, Jianlin

2018-03-01

In this study, we report the evaluation of the residue-residue contacts predicted by our three different methods in the CASP12 experiment, focusing on studying the impact of multiple sequence alignment, residue coevolution, and machine learning on contact prediction. The first method (MULTICOM-NOVEL) uses only traditional features (sequence profile, secondary structure, and solvent accessibility) with deep learning to predict contacts and serves as a baseline. The second method (MULTICOM-CONSTRUCT) uses our new alignment algorithm to generate deep multiple sequence alignment to derive coevolution-based features, which are integrated by a neural network method to predict contacts. The third method (MULTICOM-CLUSTER) is a consensus combination of the predictions of the first two methods. We evaluated our methods on 94 CASP12 domains. On a subset of 38 free-modeling domains, our methods achieved an average precision of up to 41.7% for top L/5 long-range contact predictions. The comparison of the three methods shows that the quality and effective depth of multiple sequence alignments, coevolution-based features, and machine learning integration of coevolution-based features and traditional features drive the quality of predicted protein contacts. On the full CASP12 dataset, the coevolution-based features alone can improve the average precision from 28.4% to 41.6%, and the machine learning integration of all the features further raises the precision to 56.3%, when top L/5 predicted long-range contacts are evaluated. And the correlation between the precision of contact prediction and the logarithm of the number of effective sequences in alignments is 0.66. © 2017 Wiley Periodicals, Inc.

Environmental Mycobiome Modifiers of Inflammation and Fibrosis in Systemic Sclerosis

DTIC Science & Technology

2016-09-01

TUBB), and ribosomal proteins), while others are considered specific to SSc despite trace level detection in controls. For ex- ample, multiple SSc...Strong re- activity was seen against all five proteins in SSc with only trace levels detected in controls (Fig. 3a), indicating widespread immune...sequences in SSc RNA-seq data was used to detect microbial sequences in human tissues in an unbiased, quantitative manner. Our studies suggest that

TryTransDB: A web-based resource for transport proteins in Trypanosomatidae.

PubMed

Sonar, Krushna; Kabra, Ritika; Singh, Shailza

2018-03-12

TryTransDB is a web-based resource that stores transport protein data which can be retrieved using a standalone BLAST tool. We have attempted to create an integrated database that can be a one-stop shop for the researchers working with transport proteins of Trypanosomatidae family. TryTransDB (Trypanosomatidae Transport Protein Database) is a web based comprehensive resource that can fire a BLAST search against most of the transport protein sequences (protein and nucleotide) from Trypanosomatidae family organisms. This web resource further allows to compute a phylogenetic tree by performing multiple sequence alignment (MSA) using CLUSTALW suite embedded in it. Also, cross-linking to other databases helps in gathering more information for a certain transport protein in a single website.

Template-based protein structure modeling using the RaptorX web server.

PubMed

Källberg, Morten; Wang, Haipeng; Wang, Sheng; Peng, Jian; Wang, Zhiyong; Lu, Hui; Xu, Jinbo

2012-07-19

A key challenge of modern biology is to uncover the functional role of the protein entities that compose cellular proteomes. To this end, the availability of reliable three-dimensional atomic models of proteins is often crucial. This protocol presents a community-wide web-based method using RaptorX (http://raptorx.uchicago.edu/) for protein secondary structure prediction, template-based tertiary structure modeling, alignment quality assessment and sophisticated probabilistic alignment sampling. RaptorX distinguishes itself from other servers by the quality of the alignment between a target sequence and one or multiple distantly related template proteins (especially those with sparse sequence profiles) and by a novel nonlinear scoring function and a probabilistic-consistency algorithm. Consequently, RaptorX delivers high-quality structural models for many targets with only remote templates. At present, it takes RaptorX ~35 min to finish processing a sequence of 200 amino acids. Since its official release in August 2011, RaptorX has processed ~6,000 sequences submitted by ~1,600 users from around the world.

Template-based protein structure modeling using the RaptorX web server

PubMed Central

Källberg, Morten; Wang, Haipeng; Wang, Sheng; Peng, Jian; Wang, Zhiyong; Lu, Hui; Xu, Jinbo

2016-01-01

A key challenge of modern biology is to uncover the functional role of the protein entities that compose cellular proteomes. To this end, the availability of reliable three-dimensional atomic models of proteins is often crucial. This protocol presents a community-wide web-based method using RaptorX (http://raptorx.uchicago.edu/) for protein secondary structure prediction, template-based tertiary structure modeling, alignment quality assessment and sophisticated probabilistic alignment sampling. RaptorX distinguishes itself from other servers by the quality of the alignment between a target sequence and one or multiple distantly related template proteins (especially those with sparse sequence profiles) and by a novel nonlinear scoring function and a probabilistic-consistency algorithm. Consequently, RaptorX delivers high-quality structural models for many targets with only remote templates. At present, it takes RaptorX ~35 min to finish processing a sequence of 200 amino acids. Since its official release in August 2011, RaptorX has processed ~6,000 sequences submitted by ~1,600 users from around the world. PMID:22814390

BIOPEP database and other programs for processing bioactive peptide sequences.

PubMed

Minkiewicz, Piotr; Dziuba, Jerzy; Iwaniak, Anna; Dziuba, Marta; Darewicz, Małgorzata

2008-01-01

This review presents the potential for application of computational tools in peptide science based on a sample BIOPEP database and program as well as other programs and databases available via the World Wide Web. The BIOPEP application contains a database of biologically active peptide sequences and a program enabling construction of profiles of the potential biological activity of protein fragments, calculation of quantitative descriptors as measures of the value of proteins as potential precursors of bioactive peptides, and prediction of bonds susceptible to hydrolysis by endopeptidases in a protein chain. Other bioactive and allergenic peptide sequence databases are also presented. Programs enabling the construction of binary and multiple alignments between peptide sequences, the construction of sequence motifs attributed to a given type of bioactivity, searching for potential precursors of bioactive peptides, and the prediction of sites susceptible to proteolytic cleavage in protein chains are available via the Internet as are other approaches concerning secondary structure prediction and calculation of physicochemical features based on amino acid sequence. Programs for prediction of allergenic and toxic properties have also been developed. This review explores the possibilities of cooperation between various programs.

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

A discriminative method for protein remote homology detection and fold recognition combining Top-n-grams and latent semantic analysis.

PubMed

Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

2008-12-01

Protein remote homology detection and fold recognition are central problems in bioinformatics. Currently, discriminative methods based on support vector machine (SVM) are the most effective and accurate methods for solving these problems. A key step to improve the performance of the SVM-based methods is to find a suitable representation of protein sequences. In this paper, a novel building block of proteins called Top-n-grams is presented, which contains the evolutionary information extracted from the protein sequence frequency profiles. The protein sequence frequency profiles are calculated from the multiple sequence alignments outputted by PSI-BLAST and converted into Top-n-grams. The protein sequences are transformed into fixed-dimension feature vectors by the occurrence times of each Top-n-gram. The training vectors are evaluated by SVM to train classifiers which are then used to classify the test protein sequences. We demonstrate that the prediction performance of remote homology detection and fold recognition can be improved by combining Top-n-grams and latent semantic analysis (LSA), which is an efficient feature extraction technique from natural language processing. When tested on superfamily and fold benchmarks, the method combining Top-n-grams and LSA gives significantly better results compared to related methods. The method based on Top-n-grams significantly outperforms the methods based on many other building blocks including N-grams, patterns, motifs and binary profiles. Therefore, Top-n-gram is a good building block of the protein sequences and can be widely used in many tasks of the computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the prediction of protein binding sites.

Large-Scale Concatenation cDNA Sequencing

PubMed Central

Yu, Wei; Andersson, Björn; Worley, Kim C.; Muzny, Donna M.; Ding, Yan; Liu, Wen; Ricafrente, Jennifer Y.; Wentland, Meredith A.; Lennon, Greg; Gibbs, Richard A.

1997-01-01

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching. [All 65 cDNA clone sequences described in this paper have been submitted to the GenBank data library under accession nos. U79240–U79304.] PMID:9110174

JavaScript DNA translator: DNA-aligned protein translations.

PubMed

Perry, William L

2002-12-01

There are many instances in molecular biology when it is necessary to identify ORFs in a DNA sequence. While programs exist for displaying protein translations in multiple ORFs in alignment with a DNA sequence, they are often expensive, exist as add-ons to software that must be purchased, or are only compatible with a particular operating system. JavaScript DNA Translator is a shareware application written in JavaScript, a scripting language interpreted by the Netscape Communicator and Internet Explorer Web browsers, which makes it compatible with several different operating systems. While the program uses a familiar Web page interface, it requires no connection to the Internet since calculations are performed on the user's own computer. The program analyzes one or multiple DNA sequences and generates translations in up to six reading frames aligned to a DNA sequence, in addition to displaying translations as separate sequences in FASTA format. ORFs within a reading frame can also be displayed as separate sequences. Flexible formatting options are provided, including the ability to hide ORFs below a minimum size specified by the user. The program is available free of charge at the BioTechniques Software Library (www.Biotechniques.com).

Homonuclear Hartmann-Hahn transfer with reduced relaxation losses by use of the MOCCA-XY16 multiple pulse sequence

NASA Astrophysics Data System (ADS)

Furrer, Julien; Kramer, Frank; Marino, John P.; Glaser, Steffen J.; Luy, Burkhard

2004-01-01

Homonuclear Hartmann-Hahn transfer is one of the most important building blocks in modern high-resolution NMR. It constitutes a very efficient transfer element for the assignment of proteins, nucleic acids, and oligosaccharides. Nevertheless, in macromolecules exceeding ˜10 kDa TOCSY-experiments can show decreasing sensitivity due to fast transverse relaxation processes that are active during the mixing periods. In this article we propose the MOCCA-XY16 multiple pulse sequence, originally developed for efficient TOCSY transfer through residual dipolar couplings, as a homonuclear Hartmann-Hahn sequence with improved relaxation properties. A theoretical analysis of the coherence transfer via scalar couplings and its relaxation behavior as well as experimental transfer curves for MOCCA-XY16 relative to the well-characterized DIPSI-2 multiple pulse sequence are given.

Homonuclear Hartmann-Hahn transfer with reduced relaxation losses by use of the MOCCA-XY16 multiple pulse sequence.

PubMed

Furrer, Julien; Kramer, Frank; Marino, John P; Glaser, Steffen J; Luy, Burkhard

2004-01-01

Homonuclear Hartmann-Hahn transfer is one of the most important building blocks in modern high-resolution NMR. It constitutes a very efficient transfer element for the assignment of proteins, nucleic acids, and oligosaccharides. Nevertheless, in macromolecules exceeding approximately 10 kDa TOCSY-experiments can show decreasing sensitivity due to fast transverse relaxation processes that are active during the mixing periods. In this article we propose the MOCCA-XY16 multiple pulse sequence, originally developed for efficient TOCSY transfer through residual dipolar couplings, as a homonuclear Hartmann-Hahn sequence with improved relaxation properties. A theoretical analysis of the coherence transfer via scalar couplings and its relaxation behavior as well as experimental transfer curves for MOCCA-XY16 relative to the well-characterized DIPSI-2 multiple pulse sequence are given.

PFAAT version 2.0: a tool for editing, annotating, and analyzing multiple sequence alignments.

PubMed

Caffrey, Daniel R; Dana, Paul H; Mathur, Vidhya; Ocano, Marco; Hong, Eun-Jong; Wang, Yaoyu E; Somaroo, Shyamal; Caffrey, Brian E; Potluri, Shobha; Huang, Enoch S

2007-10-11

By virtue of their shared ancestry, homologous sequences are similar in their structure and function. Consequently, multiple sequence alignments are routinely used to identify trends that relate to function. This type of analysis is particularly productive when it is combined with structural and phylogenetic analysis. Here we describe the release of PFAAT version 2.0, a tool for editing, analyzing, and annotating multiple sequence alignments. Support for multiple annotations is a key component of this release as it provides a framework for most of the new functionalities. The sequence annotations are accessible from the alignment and tree, where they are typically used to label sequences or hyperlink them to related databases. Sequence annotations can be created manually or extracted automatically from UniProt entries. Once a multiple sequence alignment is populated with sequence annotations, sequences can be easily selected and sorted through a sophisticated search dialog. The selected sequences can be further analyzed using statistical methods that explicitly model relationships between the sequence annotations and residue properties. Residue annotations are accessible from the alignment viewer and are typically used to designate binding sites or properties for a particular residue. Residue annotations are also searchable, and allow one to quickly select alignment columns for further sequence analysis, e.g. computing percent identities. Other features include: novel algorithms to compute sequence conservation, mapping conservation scores to a 3D structure in Jmol, displaying secondary structure elements, and sorting sequences by residue composition. PFAAT provides a framework whereby end-users can specify knowledge for a protein family in the form of annotation. The annotations can be combined with sophisticated analysis to test hypothesis that relate to sequence, structure and function.

PredictProtein—an open resource for online prediction of protein structural and functional features

PubMed Central

Yachdav, Guy; Kloppmann, Edda; Kajan, Laszlo; Hecht, Maximilian; Goldberg, Tatyana; Hamp, Tobias; Hönigschmid, Peter; Schafferhans, Andrea; Roos, Manfred; Bernhofer, Michael; Richter, Lothar; Ashkenazy, Haim; Punta, Marco; Schlessinger, Avner; Bromberg, Yana; Schneider, Reinhard; Vriend, Gerrit; Sander, Chris; Ben-Tal, Nir; Rost, Burkhard

2014-01-01

PredictProtein is a meta-service for sequence analysis that has been predicting structural and functional features of proteins since 1992. Queried with a protein sequence it returns: multiple sequence alignments, predicted aspects of structure (secondary structure, solvent accessibility, transmembrane helices (TMSEG) and strands, coiled-coil regions, disulfide bonds and disordered regions) and function. The service incorporates analysis methods for the identification of functional regions (ConSurf), homology-based inference of Gene Ontology terms (metastudent), comprehensive subcellular localization prediction (LocTree3), protein–protein binding sites (ISIS2), protein–polynucleotide binding sites (SomeNA) and predictions of the effect of point mutations (non-synonymous SNPs) on protein function (SNAP2). Our goal has always been to develop a system optimized to meet the demands of experimentalists not highly experienced in bioinformatics. To this end, the PredictProtein results are presented as both text and a series of intuitive, interactive and visually appealing figures. The web server and sources are available at http://ppopen.rostlab.org. PMID:24799431

Amino acid positions subject to multiple coevolutionary constraints can be robustly identified by their eigenvector network centrality scores.

PubMed

Parente, Daniel J; Ray, J Christian J; Swint-Kruse, Liskin

2015-12-01

As proteins evolve, amino acid positions key to protein structure or function are subject to mutational constraints. These positions can be detected by analyzing sequence families for amino acid conservation or for coevolution between pairs of positions. Coevolutionary scores are usually rank-ordered and thresholded to reveal the top pairwise scores, but they also can be treated as weighted networks. Here, we used network analyses to bypass a major complication of coevolution studies: For a given sequence alignment, alternative algorithms usually identify different, top pairwise scores. We reconciled results from five commonly-used, mathematically divergent algorithms (ELSC, McBASC, OMES, SCA, and ZNMI), using the LacI/GalR and 1,6-bisphosphate aldolase protein families as models. Calculations used unthresholded coevolution scores from which column-specific properties such as sequence entropy and random noise were subtracted; "central" positions were identified by calculating various network centrality scores. When compared among algorithms, network centrality methods, particularly eigenvector centrality, showed markedly better agreement than comparisons of the top pairwise scores. Positions with large centrality scores occurred at key structural locations and/or were functionally sensitive to mutations. Further, the top central positions often differed from those with top pairwise coevolution scores: instead of a few strong scores, central positions often had multiple, moderate scores. We conclude that eigenvector centrality calculations reveal a robust evolutionary pattern of constraints-detectable by divergent algorithms--that occur at key protein locations. Finally, we discuss the fact that multiple patterns coexist in evolutionary data that, together, give rise to emergent protein functions. © 2015 Wiley Periodicals, Inc.

SIBIS: a Bayesian model for inconsistent protein sequence estimation.

PubMed

Khenoussi, Walyd; Vanhoutrève, Renaud; Poch, Olivier; Thompson, Julie D

2014-09-01

The prediction of protein coding genes is a major challenge that depends on the quality of genome sequencing, the accuracy of the model used to elucidate the exonic structure of the genes and the complexity of the gene splicing process leading to different protein variants. As a consequence, today's protein databases contain a huge amount of inconsistency, due to both natural variants and sequence prediction errors. We have developed a new method, called SIBIS, to detect such inconsistencies based on the evolutionary information in multiple sequence alignments. A Bayesian framework, combined with Dirichlet mixture models, is used to estimate the probability of observing specific amino acids and to detect inconsistent or erroneous sequence segments. We evaluated the performance of SIBIS on a reference set of protein sequences with experimentally validated errors and showed that the sensitivity is significantly higher than previous methods, with only a small loss of specificity. We also assessed a large set of human sequences from the UniProt database and found evidence of inconsistency in 48% of the previously uncharacterized sequences. We conclude that the integration of quality control methods like SIBIS in automatic analysis pipelines will be critical for the robust inference of structural, functional and phylogenetic information from these sequences. Source code, implemented in C on a linux system, and the datasets of protein sequences are freely available for download at http://www.lbgi.fr/∼julie/SIBIS. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier.

PubMed

Kulmanov, Maxat; Khan, Mohammed Asif; Hoehndorf, Robert; Wren, Jonathan

2018-02-15

A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often only done rigorously for few selected model organisms. Computational function prediction approaches have been suggested to fill this gap. The functions of proteins are classified using the Gene Ontology (GO), which contains over 40 000 classes. Additionally, proteins have multiple functions, making function prediction a large-scale, multi-class, multi-label problem. We have developed a novel method to predict protein function from sequence. We use deep learning to learn features from protein sequences as well as a cross-species protein-protein interaction network. Our approach specifically outputs information in the structure of the GO and utilizes the dependencies between GO classes as background information to construct a deep learning model. We evaluate our method using the standards established by the Computational Assessment of Function Annotation (CAFA) and demonstrate a significant improvement over baseline methods such as BLAST, in particular for predicting cellular locations. Web server: http://deepgo.bio2vec.net, Source code: https://github.com/bio-ontology-research-group/deepgo. robert.hoehndorf@kaust.edu.sa. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

SATCHMO-JS: a webserver for simultaneous protein multiple sequence alignment and phylogenetic tree construction.

PubMed

Hagopian, Raffi; Davidson, John R; Datta, Ruchira S; Samad, Bushra; Jarvis, Glen R; Sjölander, Kimmen

2010-07-01

We present the jump-start simultaneous alignment and tree construction using hidden Markov models (SATCHMO-JS) web server for simultaneous estimation of protein multiple sequence alignments (MSAs) and phylogenetic trees. The server takes as input a set of sequences in FASTA format, and outputs a phylogenetic tree and MSA; these can be viewed online or downloaded from the website. SATCHMO-JS is an extension of the SATCHMO algorithm, and employs a divide-and-conquer strategy to jump-start SATCHMO at a higher point in the phylogenetic tree, reducing the computational complexity of the progressive all-versus-all HMM-HMM scoring and alignment. Results on a benchmark dataset of 983 structurally aligned pairs from the PREFAB benchmark dataset show that SATCHMO-JS provides a statistically significant improvement in alignment accuracy over MUSCLE, Multiple Alignment using Fast Fourier Transform (MAFFT), ClustalW and the original SATCHMO algorithm. The SATCHMO-JS webserver is available at http://phylogenomics.berkeley.edu/satchmo-js. The datasets used in these experiments are available for download at http://phylogenomics.berkeley.edu/satchmo-js/supplementary/.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing

PubMed Central

Sarkisova, Tatiana; Lenz, Ondřej; Přibylová, Jaroslava; Špak, Josef; Lotos, Leonidas; Beta, Christina; Katsiani, Asimina; Candresse, Thierry

2018-01-01

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species. PMID:29670059

Visualization and dissemination of multidimensional proteomics data comparing protein abundance during Caenorhabditis elegans development.

PubMed

Riffle, Michael; Merrihew, Gennifer E; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N; Noble, William S; MacCoss, Michael J

2015-11-01

Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/ . Graphical Abstract ᅟ.

Visualization and Dissemination of Multidimensional Proteomics Data Comparing Protein Abundance During Caenorhabditis elegans Development

NASA Astrophysics Data System (ADS)

Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.

2015-11-01

Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data

PubMed Central

Dotu, Ivan; Adamson, Scott I.; Coleman, Benjamin; Fournier, Cyril; Ricart-Altimiras, Emma; Eyras, Eduardo

2018-01-01

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC. PMID:29596423

Evol and ProDy for bridging protein sequence evolution and structural dynamics.

PubMed

Bakan, Ahmet; Dutta, Anindita; Mao, Wenzhi; Liu, Ying; Chennubhotla, Chakra; Lezon, Timothy R; Bahar, Ivet

2014-09-15

Correlations between sequence evolution and structural dynamics are of utmost importance in understanding the molecular mechanisms of function and their evolution. We have integrated Evol, a new package for fast and efficient comparative analysis of evolutionary patterns and conformational dynamics, into ProDy, a computational toolbox designed for inferring protein dynamics from experimental and theoretical data. Using information-theoretic approaches, Evol coanalyzes conservation and coevolution profiles extracted from multiple sequence alignments of protein families with their inferred dynamics. ProDy and Evol are open-source and freely available under MIT License from http://prody.csb.pitt.edu/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

fLPS: Fast discovery of compositional biases for the protein universe.

PubMed

Harrison, Paul M

2017-11-13

Proteins often contain regions that are compositionally biased (CB), i.e., they are made from a small subset of amino-acid residue types. These CB regions can be functionally important, e.g., the prion-forming and prion-like regions that are rich in asparagine and glutamine residues. Here I report a new program fLPS that can rapidly annotate CB regions. It discovers both single-residue and multiple-residue biases. It works through a process of probability minimization. First, contigs are constructed for each amino-acid type out of sequence windows with a low degree of bias; second, these contigs are searched exhaustively for low-probability subsequences (LPSs); third, such LPSs are iteratively assessed for merger into possible multiple-residue biases. At each of these stages, efficiency measures are taken to avoid or delay probability calculations unless/until they are necessary. On a current desktop workstation, the fLPS algorithm can annotate the biased regions of the yeast proteome (>5700 sequences) in <1 s, and of the whole current TrEMBL database (>65 million sequences) in as little as ~1 h, which is >2 times faster than the commonly used program SEG, using default parameters. fLPS discovers both shorter CB regions (of the sort that are often termed 'low-complexity sequence'), and milder biases that may only be detectable over long tracts of sequence. fLPS can readily handle very large protein data sets, such as might come from metagenomics projects. It is useful in searching for proteins with similar CB regions, and for making functional inferences about CB regions for a protein of interest. The fLPS package is available from: http://biology.mcgill.ca/faculty/harrison/flps.html , or https://github.com/pmharrison/flps , or is a supplement to this article.

PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

PubMed

Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

2016-07-08

The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

MOCASSIN-prot: a multi-objective clustering approach for protein similarity networks.

PubMed

Keel, Brittney N; Deng, Bo; Moriyama, Etsuko N

2018-04-15

Proteins often include multiple conserved domains. Various evolutionary events including duplication and loss of domains, domain shuffling, as well as sequence divergence contribute to generating complexities in protein structures, and consequently, in their functions. The evolutionary history of proteins is hence best modeled through networks that incorporate information both from the sequence divergence and the domain content. Here, a game-theoretic approach proposed for protein network construction is adapted into the framework of multi-objective optimization, and extended to incorporate clustering refinement procedure. The new method, MOCASSIN-prot, was applied to cluster multi-domain proteins from ten genomes. The performance of MOCASSIN-prot was compared against two protein clustering methods, Markov clustering (TRIBE-MCL) and spectral clustering (SCPS). We showed that compared to these two methods, MOCASSIN-prot, which uses both domain composition and quantitative sequence similarity information, generates fewer false positives. It achieves more functionally coherent protein clusters and better differentiates protein families. MOCASSIN-prot, implemented in Perl and Matlab, is freely available at http://bioinfolab.unl.edu/emlab/MOCASSINprot. emoriyama2@unl.edu. Supplementary data are available at Bioinformatics online.

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation.

PubMed

Ehrmann, M A; Vogel, R E

2001-11-01

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.

Self-sequencing of amino acids and origins of polyfunctional protocells

NASA Technical Reports Server (NTRS)

Fox, S. W.

1984-01-01

The role of proteins in the origin of living things is discussed. It has been experimentally established that amino acids can sequence themselves under simulated geological conditions with highly nonrandom products which accordingly contain diverse information. Multiple copies of each type of macromolecule are formed, resulting in greater power for any protoenzymic molecule than would accrue from a single copy of each type. Thermal proteins are readily incorporated into laboratory protocells. The experimental evidence for original polyfunctional protocells is discussed.

Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

PubMed

Barnard, G F; Staniunas, R J; Puder, M; Steele, G D; Chen, L B

1994-08-02

Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

Incorporating information on predicted solvent accessibility to the co-evolution-based study of protein interactions.

PubMed

Ochoa, David; García-Gutiérrez, Ponciano; Juan, David; Valencia, Alfonso; Pazos, Florencio

2013-01-27

A widespread family of methods for studying and predicting protein interactions using sequence information is based on co-evolution, quantified as similarity of phylogenetic trees. Part of the co-evolution observed between interacting proteins could be due to co-adaptation caused by inter-protein contacts. In this case, the co-evolution is expected to be more evident when evaluated on the surface of the proteins or the internal layers close to it. In this work we study the effect of incorporating information on predicted solvent accessibility to three methods for predicting protein interactions based on similarity of phylogenetic trees. We evaluate the performance of these methods in predicting different types of protein associations when trees based on positions with different characteristics of predicted accessibility are used as input. We found that predicted accessibility improves the results of two recent versions of the mirrortree methodology in predicting direct binary physical interactions, while it neither improves these methods, nor the original mirrortree method, in predicting other types of interactions. That improvement comes at no cost in terms of applicability since accessibility can be predicted for any sequence. We also found that predictions of protein-protein interactions are improved when multiple sequence alignments with a richer representation of sequences (including paralogs) are incorporated in the accessibility prediction.

Common Amino Acid Subsequences in a Universal Proteome—Relevance for Food Science

PubMed Central

Minkiewicz, Piotr; Darewicz, Małgorzata; Iwaniak, Anna; Sokołowska, Jolanta; Starowicz, Piotr; Bucholska, Justyna; Hrynkiewicz, Monika

2015-01-01

A common subsequence is a fragment of the amino acid chain that occurs in more than one protein. Common subsequences may be an object of interest for food scientists as biologically active peptides, epitopes, and/or protein markers that are used in comparative proteomics. An individual bioactive fragment, in particular the shortest fragment containing two or three amino acid residues, may occur in many protein sequences. An individual linear epitope may also be present in multiple sequences of precursor proteins. Although recent recommendations for prediction of allergenicity and cross-reactivity include not only sequence identity, but also similarities in secondary and tertiary structures surrounding the common fragment, local sequence identity may be used to screen protein sequence databases for potential allergens in silico. The main weakness of the screening process is that it overlooks allergens and cross-reactivity cases without identical fragments corresponding to linear epitopes. A single peptide may also serve as a marker of a group of allergens that belong to the same family and, possibly, reveal cross-reactivity. This review article discusses the benefits for food scientists that follow from the common subsequences concept. PMID:26340620

HAlign-II: efficient ultra-large multiple sequence alignment and phylogenetic tree reconstruction with distributed and parallel computing.

PubMed

Wan, Shixiang; Zou, Quan

2017-01-01

Multiple sequence alignment (MSA) plays a key role in biological sequence analyses, especially in phylogenetic tree construction. Extreme increase in next-generation sequencing results in shortage of efficient ultra-large biological sequence alignment approaches for coping with different sequence types. Distributed and parallel computing represents a crucial technique for accelerating ultra-large (e.g. files more than 1 GB) sequence analyses. Based on HAlign and Spark distributed computing system, we implement a highly cost-efficient and time-efficient HAlign-II tool to address ultra-large multiple biological sequence alignment and phylogenetic tree construction. The experiments in the DNA and protein large scale data sets, which are more than 1GB files, showed that HAlign II could save time and space. It outperformed the current software tools. HAlign-II can efficiently carry out MSA and construct phylogenetic trees with ultra-large numbers of biological sequences. HAlign-II shows extremely high memory efficiency and scales well with increases in computing resource. THAlign-II provides a user-friendly web server based on our distributed computing infrastructure. HAlign-II with open-source codes and datasets was established at http://lab.malab.cn/soft/halign.

Structure of a Trypanosoma Brucei Alpha/Beta--Hydrolase Fold Protein With Unknown Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merritt, E.A.; Holmes, M.; Buckner, F.S.

2009-05-26

The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 {angstrom} using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the {alpha}/{beta}-hydrolase fold family. Structural superposition onto representative {alpha}/{beta}-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similaritymore » at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands {beta}6 and {beta}7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family.« less

Protein Sequence Annotation Tool (PSAT): A centralized web-based meta-server for high-throughput sequence annotations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Elo; Huang, Amy; Cadag, Eithon

In this study, we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resultingmore » functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. Lastly, PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequencebased genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.« less

Protein Sequence Annotation Tool (PSAT): A centralized web-based meta-server for high-throughput sequence annotations

DOE PAGES

Leung, Elo; Huang, Amy; Cadag, Eithon; ...

2016-01-20

In this study, we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resultingmore » functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. Lastly, PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequencebased genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.« less

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

PubMed Central

Celorio-Mancera, Maria de la Paz; Courtiade, Juliette; Muck, Alexander; Heckel, David G.; Musser, Richard O.; Vogel, Heiko

2011-01-01

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function. PMID:22046331

Food-derived immunomodulatory peptides.

PubMed

Santiago-López, Lourdes; Hernández-Mendoza, Adrián; Vallejo-Cordoba, Belinda; Mata-Haro, Verónica; González-Córdova, Aarón F

2016-08-01

Food proteins contain specific amino acid sequences within their structures that may positively impact bodily functions and have multiple immunomodulatory effects. The functional properties of these specific sequences, also referred to as bioactive peptides, are revealed only after the degradation of native proteins during digestion processes. Currently, milk proteins have been the most explored source of bioactive peptides, which presents an interesting opportunity for the dairy industry. However, plant- and animal-derived proteins have also been shown to be important sources of bioactive peptides. This review summarizes the in vitro and in vivo evidence of the role of various food proteins as sources of immunomodulatory peptides and discusses the possible pathways involving these properties. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

The Reference Genome of the Halophytic Plant Eutrema salsugineum

PubMed Central

Yang, Ruolin; Jarvis, David E.; Chen, Hao; Beilstein, Mark A.; Grimwood, Jane; Jenkins, Jerry; Shu, ShengQiang; Prochnik, Simon; Xin, Mingming; Ma, Chuang; Schmutz, Jeremy; Wing, Rod A.; Mitchell-Olds, Thomas; Schumaker, Karen S.; Wang, Xiangfeng

2013-01-01

Halophytes are plants that can naturally tolerate high concentrations of salt in the soil, and their tolerance to salt stress may occur through various evolutionary and molecular mechanisms. Eutrema salsugineum is a halophytic species in the Brassicaceae that can naturally tolerate multiple types of abiotic stresses that typically limit crop productivity, including extreme salinity and cold. It has been widely used as a laboratorial model for stress biology research in plants. Here, we present the reference genome sequence (241 Mb) of E. salsugineum at 8× coverage sequenced using the traditional Sanger sequencing-based approach with comparison to its close relative Arabidopsis thaliana. The E. salsugineum genome contains 26,531 protein-coding genes and 51.4% of its genome is composed of repetitive sequences that mostly reside in pericentromeric regions. Comparative analyses of the genome structures, protein-coding genes, microRNAs, stress-related pathways, and estimated translation efficiency of proteins between E. salsugineum and A. thaliana suggest that halophyte adaptation to environmental stresses may occur via a global network adjustment of multiple regulatory mechanisms. The E. salsugineum genome provides a resource to identify naturally occurring genetic alterations contributing to the adaptation of halophytic plants to salinity and that might be bioengineered in related crop species. PMID:23518688

Integrated databanks access and sequence/structure analysis services at the PBIL.

PubMed

Perrière, Guy; Combet, Christophe; Penel, Simon; Blanchet, Christophe; Thioulouse, Jean; Geourjon, Christophe; Grassot, Julien; Charavay, Céline; Gouy, Manolo; Duret, Laurent; Deléage, Gilbert

2003-07-01

The World Wide Web server of the PBIL (Pôle Bioinformatique Lyonnais) provides on-line access to sequence databanks and to many tools of nucleic acid and protein sequence analyses. This server allows to query nucleotide sequence banks in the EMBL and GenBank formats and protein sequence banks in the SWISS-PROT and PIR formats. The query engine on which our data bank access is based is the ACNUC system. It allows the possibility to build complex queries to access functional zones of biological interest and to retrieve large sequence sets. Of special interest are the unique features provided by this system to query the data banks of gene families developed at the PBIL. The server also provides access to a wide range of sequence analysis methods: similarity search programs, multiple alignments, protein structure prediction and multivariate statistics. An originality of this server is the integration of these two aspects: sequence retrieval and sequence analysis. Indeed, thanks to the introduction of re-usable lists, it is possible to perform treatments on large sets of data. The PBIL server can be reached at: http://pbil.univ-lyon1.fr.

Comparative Analysis and Distribution of Omega-3 lcPUFA Biosynthesis Genes in Marine Molluscs

PubMed Central

Surm, Joachim M.; Prentis, Peter J.; Pavasovic, Ana

2015-01-01

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs. PMID:26308548

Sequence, structure and function relationships in flaviviruses as assessed by evolutive aspects of its conserved non-structural protein domains.

PubMed

da Fonseca, Néli José; Lima Afonso, Marcelo Querino; Pedersolli, Natan Gonçalves; de Oliveira, Lucas Carrijo; Andrade, Dhiego Souto; Bleicher, Lucas

2017-10-28

Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative In silico Study of Sex-Determining Region Y (SRY) Protein Sequences Involved in Sex-Determining.

PubMed

Vakili Azghandi, Masoume; Nasiri, Mohammadreza; Shamsa, Ali; Jalali, Mohsen; Shariati, Mohammad Mahdi

2016-04-01

The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares. The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

Protein location prediction using atomic composition and global features of the amino acid sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherian, Betsy Sheena, E-mail: betsy.skb@gmail.com; Nair, Achuthsankar S.

2010-01-22

Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectivelymore » used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.« less

A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3

PubMed Central

Dietmann, Sabine; Park, Jong; Notredame, Cedric; Heger, Andreas; Lappe, Michael; Holm, Liisa

2001-01-01

The Dali Domain Dictionary (http://www.ebi.ac.uk/dali/domain) is a numerical taxonomy of all known structures in the Protein Data Bank (PDB). The taxonomy is derived fully automatically from measurements of structural, functional and sequence similarities. Here, we report the extension of the classification to match the traditional four hierarchical levels corresponding to: (i) supersecondary structural motifs (attractors in fold space), (ii) the topology of globular domains (fold types), (iii) remote homologues (functional families) and (iv) homologues with sequence identity above 25% (sequence families). The computational definitions of attractors and functional families are new. In September 2000, the Dali classification contained 10 531 PDB entries comprising 17 101 chains, which were partitioned into five attractor regions, 1375 fold types, 2582 functional families and 3724 domain sequence families. Sequence families were further associated with 99 582 unique homologous sequences in the HSSP database, which increases the number of effectively known structures several-fold. The resulting database contains the description of protein domain architecture, the definition of structural neighbours around each known structure, the definition of structurally conserved cores and a comprehensive library of explicit multiple alignments of distantly related protein families. PMID:11125048

Automated design of degenerate codon libraries.

PubMed

Mena, Marco A; Daugherty, Patrick S

2005-12-01

Degenerate codon libraries are frequently used in protein engineering and evolution studies but are often limited to targeting a small number of positions to adequately limit the search space. To mitigate this, codon degeneracy can be limited using heuristics or previous knowledge of the targeted positions. To automate design of libraries given a set of amino acid sequences, an algorithm (LibDesign) was developed that generates a set of possible degenerate codon libraries, their resulting size, and their score relative to a user-defined scoring function. A gene library of a specified size can then be constructed that is representative of the given amino acid distribution or that includes specific sequences or combinations thereof. LibDesign provides a new tool for automated design of high-quality protein libraries that more effectively harness existing sequence-structure information derived from multiple sequence alignment or computational protein design data.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

PubMed Central

Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

2016-01-01

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

Predicting RNA-protein binding sites and motifs through combining local and global deep convolutional neural networks.

PubMed

Pan, Xiaoyong; Shen, Hong-Bin

2018-05-02

RNA-binding proteins (RBPs) take over 5∼10% of the eukaryotic proteome and play key roles in many biological processes, e.g. gene regulation. Experimental detection of RBP binding sites is still time-intensive and high-costly. Instead, computational prediction of the RBP binding sites using pattern learned from existing annotation knowledge is a fast approach. From the biological point of view, the local structure context derived from local sequences will be recognized by specific RBPs. However, in computational modeling using deep learning, to our best knowledge, only global representations of entire RNA sequences are employed. So far, the local sequence information is ignored in the deep model construction process. In this study, we present a computational method iDeepE to predict RNA-protein binding sites from RNA sequences by combining global and local convolutional neural networks (CNNs). For the global CNN, we pad the RNA sequences into the same length. For the local CNN, we split a RNA sequence into multiple overlapping fixed-length subsequences, where each subsequence is a signal channel of the whole sequence. Next, we train deep CNNs for multiple subsequences and the padded sequences to learn high-level features, respectively. Finally, the outputs from local and global CNNs are combined to improve the prediction. iDeepE demonstrates a better performance over state-of-the-art methods on two large-scale datasets derived from CLIP-seq. We also find that the local CNN run 1.8 times faster than the global CNN with comparable performance when using GPUs. Our results show that iDeepE has captured experimentally verified binding motifs. https://github.com/xypan1232/iDeepE. xypan172436@gmail.com or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online.

The Histone Database: an integrated resource for histones and histone fold-containing proteins

PubMed Central

Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

2011-01-01

Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

RStrucFam: a web server to associate structure and cognate RNA for RNA-binding proteins from sequence information.

PubMed

Ghosh, Pritha; Mathew, Oommen K; Sowdhamini, Ramanathan

2016-10-07

RNA-binding proteins (RBPs) interact with their cognate RNA(s) to form large biomolecular assemblies. They are versatile in their functionality and are involved in a myriad of processes inside the cell. RBPs with similar structural features and common biological functions are grouped together into families and superfamilies. It will be useful to obtain an early understanding and association of RNA-binding property of sequences of gene products. Here, we report a web server, RStrucFam, to predict the structure, type of cognate RNA(s) and function(s) of proteins, where possible, from mere sequence information. The web server employs Hidden Markov Model scan (hmmscan) to enable association to a back-end database of structural and sequence families. The database (HMMRBP) comprises of 437 HMMs of RBP families of known structure that have been generated using structure-based sequence alignments and 746 sequence-centric RBP family HMMs. The input protein sequence is associated with structural or sequence domain families, if structure or sequence signatures exist. In case of association of the protein with a family of known structures, output features like, multiple structure-based sequence alignment (MSSA) of the query with all others members of that family is provided. Further, cognate RNA partner(s) for that protein, Gene Ontology (GO) annotations, if any and a homology model of the protein can be obtained. The users can also browse through the database for details pertaining to each family, protein or RNA and their related information based on keyword search or RNA motif search. RStrucFam is a web server that exploits structurally conserved features of RBPs, derived from known family members and imprinted in mathematical profiles, to predict putative RBPs from sequence information. Proteins that fail to associate with such structure-centric families are further queried against the sequence-centric RBP family HMMs in the HMMRBP database. Further, all other essential information pertaining to an RBP, like overall function annotations, are provided. The web server can be accessed at the following link: http://caps.ncbs.res.in/rstrucfam .

RPG: the Ribosomal Protein Gene database.

PubMed

Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

2004-01-01

RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

RPG: the Ribosomal Protein Gene database

PubMed Central

Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

2004-01-01

RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes. PMID:14681386

A Method for WD40 Repeat Detection and Secondary Structure Prediction

PubMed Central

Wang, Yang; Jiang, Fan; Zhuo, Zhu; Wu, Xian-Hui; Wu, Yun-Dong

2013-01-01

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar β-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction. PMID:23776530

Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

PubMed Central

Cai, Yongping; Lin, Yi

2013-01-01

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

Molecular Cloning of Drebrin: Progress and Perspectives.

PubMed

Kojima, Nobuhiko

2017-01-01

Chicken drebrin isoforms were first identified in the optic tectum of developing brain. Although the time course of protein expression was different in each drebrin isoform, the similarity between their protein structures was suggested by biochemical analysis of purified protein. To determine their protein structures, the cloning of drebrin cDNAs was conducted. Comparison between the cDNA sequences shows that all drebrin cDNAs are identical except that the internal insertion sequences are present or absent in their sequences. Chicken drebrin are now classified into three isoforms, namely, drebrins E1, E2, and A. Genomic cloning demonstrated that the three isoforms are generated by an alternative splicing of individual exons encoding the insertion sequences from single drebrin gene. The mechanism should be precisely regulated in cell-type-specific and developmental stage-specific fashion. Drebrin protein, which is well conserved in various vertebrate species, although mammalian drebrin has only two isoforms, namely, drebrin E and drebrin A, is different from chicken drebrin that has three isoforms. Drebrin belongs to an actin-depolymerizing factor homology (ADF-H) domain protein family. Besides the ADF-H domain, drebrin has other domains, including the actin-binding domain and Homer-binding motifs. Diversity of protein isoform and multiple domains of drebrin could interact differentially with the actin cytoskeleton and other intracellular proteins and regulate diverse cellular processes.

Informational structure of genetic sequences and nature of gene splicing

NASA Astrophysics Data System (ADS)

Trifonov, E. N.

1991-10-01

Only about 1/20 of DNA of higher organisms codes for proteins, by means of classical triplet code. The rest of DNA sequences is largely silent, with unclear functions, if any. The triplet code is not the only code (message) carried by the sequences. There are three levels of molecular communication, where the same sequence ``talks'' to various bimolecules, while having, respectively, three different appearances: DNA, RNA and protein. Since the molecular structures and, hence, sequence specific preferences of these are substantially different, the original DNA sequence has to carry simultaneously three types of sequence patterns (codes, messages), thus, being a composite structure in which one had the same letter (nucleotide) is frequently involved in several overlapping codes of different nature. This multiplicity and overlapping of the codes is a unique feature of the Gnomic, language of genetic sequences. The coexisting codes have to be degenerate in various degrees to allow an optimal and concerted performance of all the encoded functions. There is an obvious conflict between the best possible performance of a given function and necessity to compromise the quality of a given sequence pattern in favor of other patterns. It appears that the major role of various changes in the sequences on their ``ontogenetic'' way from DNA to RNA to protein, like RNA editing and splicing, or protein post-translational modifications is to resolve such conflicts. New data are presented strongly indicating that the gene splicing is such a device to resolve the conflict between the code of DNA folding in chromatin and the triplet code for protein synthesis.

Discovering Sequence Motifs with Arbitrary Insertions and Deletions

PubMed Central

Frith, Martin C.; Saunders, Neil F. W.; Kobe, Bostjan; Bailey, Timothy L.

2008-01-01

Biology is encoded in molecular sequences: deciphering this encoding remains a grand scientific challenge. Functional regions of DNA, RNA, and protein sequences often exhibit characteristic but subtle motifs; thus, computational discovery of motifs in sequences is a fundamental and much-studied problem. However, most current algorithms do not allow for insertions or deletions (indels) within motifs, and the few that do have other limitations. We present a method, GLAM2 (Gapped Local Alignment of Motifs), for discovering motifs allowing indels in a fully general manner, and a companion method GLAM2SCAN for searching sequence databases using such motifs. glam2 is a generalization of the gapless Gibbs sampling algorithm. It re-discovers variable-width protein motifs from the PROSITE database significantly more accurately than the alternative methods PRATT and SAM-T2K. Furthermore, it usefully refines protein motifs from the ELM database: in some cases, the refined motifs make orders of magnitude fewer overpredictions than the original ELM regular expressions. GLAM2 performs respectably on the BAliBASE multiple alignment benchmark, and may be superior to leading multiple alignment methods for “motif-like” alignments with N- and C-terminal extensions. Finally, we demonstrate the use of GLAM2 to discover protein kinase substrate motifs and a gapped DNA motif for the LIM-only transcriptional regulatory complex: using GLAM2SCAN, we identify promising targets for the latter. GLAM2 is especially promising for short protein motifs, and it should improve our ability to identify the protein cleavage sites, interaction sites, post-translational modification attachment sites, etc., that underlie much of biology. It may be equally useful for arbitrarily gapped motifs in DNA and RNA, although fewer examples of such motifs are known at present. GLAM2 is public domain software, available for download at http://bioinformatics.org.au/glam2. PMID:18437229

Protein contact prediction using patterns of correlation.

PubMed

Hamilton, Nicholas; Burrage, Kevin; Ragan, Mark A; Huber, Thomas

2004-09-01

We describe a new method for using neural networks to predict residue contact pairs in a protein. The main inputs to the neural network are a set of 25 measures of correlated mutation between all pairs of residues in two "windows" of size 5 centered on the residues of interest. While the individual pair-wise correlations are a relatively weak predictor of contact, by training the network on windows of correlation the accuracy of prediction is significantly improved. The neural network is trained on a set of 100 proteins and then tested on a disjoint set of 1033 proteins of known structure. An average predictive accuracy of 21.7% is obtained taking the best L/2 predictions for each protein, where L is the sequence length. Taking the best L/10 predictions gives an average accuracy of 30.7%. The predictor is also tested on a set of 59 proteins from the CASP5 experiment. The accuracy is found to be relatively consistent across different sequence lengths, but to vary widely according to the secondary structure. Predictive accuracy is also found to improve by using multiple sequence alignments containing many sequences to calculate the correlations. Copyright 2004 Wiley-Liss, Inc.

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

A Bayesian Sampler for Optimization of Protein Domain Hierarchies

PubMed Central

2014-01-01

Abstract The process of identifying and modeling functionally divergent subgroups for a specific protein domain class and arranging these subgroups hierarchically has, thus far, largely been done via manual curation. How to accomplish this automatically and optimally is an unsolved statistical and algorithmic problem that is addressed here via Markov chain Monte Carlo sampling. Taking as input a (typically very large) multiple-sequence alignment, the sampler creates and optimizes a hierarchy by adding and deleting leaf nodes, by moving nodes and subtrees up and down the hierarchy, by inserting or deleting internal nodes, and by redefining the sequences and conserved patterns associated with each node. All such operations are based on a probability distribution that models the conserved and divergent patterns defining each subgroup. When we view these patterns as sequence determinants of protein function, each node or subtree in such a hierarchy corresponds to a subgroup of sequences with similar biological properties. The sampler can be applied either de novo or to an existing hierarchy. When applied to 60 protein domains from multiple starting points in this way, it converged on similar solutions with nearly identical log-likelihood ratio scores, suggesting that it typically finds the optimal peak in the posterior probability distribution. Similarities and differences between independently generated, nearly optimal hierarchies for a given domain help distinguish robust from statistically uncertain features. Thus, a future application of the sampler is to provide confidence measures for various features of a domain hierarchy. PMID:24494927

Rapid detection, classification and accurate alignment of up to a million or more related protein sequences.

PubMed

Neuwald, Andrew F

2009-08-01

The patterns of sequence similarity and divergence present within functionally diverse, evolutionarily related proteins contain implicit information about corresponding biochemical similarities and differences. A first step toward accessing such information is to statistically analyze these patterns, which, in turn, requires that one first identify and accurately align a very large set of protein sequences. Ideally, the set should include many distantly related, functionally divergent subgroups. Because it is extremely difficult, if not impossible for fully automated methods to align such sequences correctly, researchers often resort to manual curation based on detailed structural and biochemical information. However, multiply-aligning vast numbers of sequences in this way is clearly impractical. This problem is addressed using Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS). The MAPGAPS program uses a set of multiply-aligned profiles both as a query to detect and classify related sequences and as a template to multiply-align the sequences. It relies on Karlin-Altschul statistics for sensitivity and on PSI-BLAST (and other) heuristics for speed. Using as input a carefully curated multiple-profile alignment for P-loop GTPases, MAPGAPS correctly aligned weakly conserved sequence motifs within 33 distantly related GTPases of known structure. By comparison, the sequence- and structurally based alignment methods hmmalign and PROMALS3D misaligned at least 11 and 23 of these regions, respectively. When applied to a dataset of 65 million protein sequences, MAPGAPS identified, classified and aligned (with comparable accuracy) nearly half a million putative P-loop GTPase sequences. A C++ implementation of MAPGAPS is available at http://mapgaps.igs.umaryland.edu. Supplementary data are available at Bioinformatics online.

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

PubMed

Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2017-09-01

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases.

PubMed

Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

2007-10-18

Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and code examples are available at http://www.ebi.ac.uk/Tools/picr.

The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases

PubMed Central

Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

2007-01-01

Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and code examples are available at . PMID:17945017

Building toy models of proteins using coevolutionary information

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Raghunathan, Mohit; Onuchic, Jose

2015-03-01

Recent developments in global statistical methodologies have advanced the analysis of large collections of protein sequences for coevolutionary information. Coevolution between amino acids in a protein arises from compensatory mutations that are needed to maintain the stability or function of a protein over the course of evolution. This gives rise to quantifiable correlations between amino acid positions within the multiple sequence alignment of a protein family. Here, we use Direct Coupling Analysis (DCA) to infer a Potts model Hamiltonian governing the correlated mutations in a protein family to obtain the sequence-dependent interaction energies of a toy protein model. We demonstrate that this methodology predicts residue-residue interaction energies that are consistent with experimental mutational changes in protein stabilities as well as other computational methodologies. Furthermore, we demonstrate with several examples that DCA could be used to construct a structure-based model that quantitatively agrees with experimental data on folding mechanisms. This work serves as a potential framework for generating models of proteins that are enriched by evolutionary data that can potentially be used to engineer key functional motions and interactions in protein systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1427654).

A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins.

PubMed

Sawle, Lucas; Ghosh, Kingshuk

2015-08-28

A general formalism to compute configurational properties of proteins and other heteropolymers with an arbitrary sequence of charges and non-uniform excluded volume interaction is presented. A variational approach is utilized to predict average distance between any two monomers in the chain. The presented analytical model, for the first time, explicitly incorporates the role of sequence charge distribution to determine relative sizes between two sequences that vary not only in total charge composition but also in charge decoration (even when charge composition is fixed). Furthermore, the formalism is general enough to allow variation in excluded volume interactions between two monomers. Model predictions are benchmarked against the all-atom Monte Carlo studies of Das and Pappu [Proc. Natl. Acad. Sci. U. S. A. 110, 13392 (2013)] for 30 different synthetic sequences of polyampholytes. These sequences possess an equal number of glutamic acid (E) and lysine (K) residues but differ in the patterning within the sequence. Without any fit parameter, the model captures the strong sequence dependence of the simulated values of the radius of gyration with a correlation coefficient of R(2) = 0.9. The model is then applied to real proteins to compare the unfolded state dimensions of 540 orthologous pairs of thermophilic and mesophilic proteins. The excluded volume parameters are assumed similar under denatured conditions, and only electrostatic effects encoded in the sequence are accounted for. With these assumptions, thermophilic proteins are found-with high statistical significance-to have more compact disordered ensemble compared to their mesophilic counterparts. The method presented here, due to its analytical nature, is capable of making such high throughput analysis of multiple proteins and will have broad applications in proteomic studies as well as in other heteropolymeric systems.

Adaptive Local Realignment of Protein Sequences.

PubMed

DeBlasio, Dan; Kececioglu, John

2018-06-11

While mutation rates can vary markedly over the residues of a protein, multiple sequence alignment tools typically use the same values for their scoring-function parameters across a protein's entire length. We present a new approach, called adaptive local realignment, that in contrast automatically adapts to the diversity of mutation rates along protein sequences. This builds upon a recent technique known as parameter advising, which finds global parameter settings for an aligner, to now adaptively find local settings. Our approach in essence identifies local regions with low estimated accuracy, constructs a set of candidate realignments using a carefully-chosen collection of parameter settings, and replaces the region if a realignment has higher estimated accuracy. This new method of local parameter advising, when combined with prior methods for global advising, boosts alignment accuracy as much as 26% over the best default setting on hard-to-align protein benchmarks, and by 6.4% over global advising alone. Adaptive local realignment has been implemented within the Opal aligner using the Facet accuracy estimator.

Automatic Prediction of Protein 3D Structures by Probabilistic Multi-template Homology Modeling.

PubMed

Meier, Armin; Söding, Johannes

2015-10-01

Homology modeling predicts the 3D structure of a query protein based on the sequence alignment with one or more template proteins of known structure. Its great importance for biological research is owed to its speed, simplicity, reliability and wide applicability, covering more than half of the residues in protein sequence space. Although multiple templates have been shown to generally increase model quality over single templates, the information from multiple templates has so far been combined using empirically motivated, heuristic approaches. We present here a rigorous statistical framework for multi-template homology modeling. First, we find that the query proteins' atomic distance restraints can be accurately described by two-component Gaussian mixtures. This insight allowed us to apply the standard laws of probability theory to combine restraints from multiple templates. Second, we derive theoretically optimal weights to correct for the redundancy among related templates. Third, a heuristic template selection strategy is proposed. We improve the average GDT-ha model quality score by 11% over single template modeling and by 6.5% over a conventional multi-template approach on a set of 1000 query proteins. Robustness with respect to wrong constraints is likewise improved. We have integrated our multi-template modeling approach with the popular MODELLER homology modeling software in our free HHpred server http://toolkit.tuebingen.mpg.de/hhpred and also offer open source software for running MODELLER with the new restraints at https://bitbucket.org/soedinglab/hh-suite.

Differentiated evolutionary relationships among chordates from comparative alignments of multiple sequences of MyoD and MyoG myogenic regulatory factors.

PubMed

Oliani, L C; Lidani, K C F; Gabriel, J E

2015-10-16

MyoD and MyoG are transcription factors that have essential roles in myogenic lineage determination and muscle differentiation. The purpose of this study was to compare multiple amino acid sequences of myogenic regulatory proteins to infer evolutionary relationships among chordates. Protein sequences from Mus musculus (P10085 and P12979), human Homo sapiens (P15172 and P15173), bovine Bos taurus (Q7YS82 and Q7YS81), wild pig Sus scrofa (P49811 and P49812), quail Coturnix coturnix (P21572 and P34060), chicken Gallus gallus (P16075 and P17920), rat Rattus norvegicus (Q02346 and P20428), domestic water buffalo Bubalus bubalis (D2SP11 and A7L034), and sheep Ovis aries (Q90477 and D3YKV7) were searched from a non-redundant protein sequence database UniProtKB/Swiss-Prot, and subsequently analyzed using the Mega6.0 software. MyoD evolutionary analyses revealed the presence of three main clusters with all mammals branched in one cluster, members of the order Rodentia (mouse and rat) in a second branch linked to the first, and birds of the order Galliformes (chicken and quail) remaining isolated in a third. MyoG evolutionary analyses aligned sequences in two main clusters, all mammalian specimens grouped in different sub-branches, and birds clustered in a second branch. These analyses suggest that the evolution of MyoD and MyoG was driven by different pathways.

Sequence identity and antigenic cross-reactivity of white face hornet venom allergen, also a hyaluronidase, with other proteins.

PubMed

Lu, G; Kochoumian, L; King, T P

1995-03-03

White face hornet (Dolichovespula maculata) venom has three known protein allergens which induce IgE response in susceptible people. They are antigen 5, phospholipase A1, and hyaluronidase, also known as Dol m 5, 1, and 2, respectively. We have cloned Dol m 2, a protein of 331 residues. When expressed in bacteria, a mixture of recombinant Dol m 2 and its fragments was obtained. The fragments were apparently generated by proteolysis of a Met-Met bond at residue 122, as they were not observed for a Dol m 2 mutant with a Leu-Met bond. Dol m 2 has 56% sequence identity with the honey bee venom allergen hyaluronidase and 27% identity with PH-20, a human sperm protein with hyaluronidase activity. A common feature of hornet venom allergens is their sequence identity with other proteins in our environment. We showed previously the sequence identity of Dol m 5 with a plant protein and a mammalian testis protein and of Dol m 1 with mammalian lipases. In BALB/c mice, Dol m 2 and bee hyaluronidase showed cross-reactivity at both antibody and T cell levels. These findings are relevant to some patients' multiple sensitivity to hornet and bee stings.

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase.

PubMed

Zemla, Adam T; Lang, Dorothy M; Kostova, Tanya; Andino, Raul; Ecale Zhou, Carol L

2011-06-02

Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory--still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could help overcome these difficulties by facilitating the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. StralSV is provided as a web service at http://proteinmodel.org/AS2TS/STRALSV/.

Protein Structure and Function Prediction Using I-TASSER

PubMed Central

Yang, Jianyi; Zhang, Yang

2016-01-01

I-TASSER is a hierarchical protocol for automated protein structure prediction and structure-based function annotation. Starting from the amino acid sequence of target proteins, I-TASSER first generates full-length atomic structural models from multiple threading alignments and iterative structural assembly simulations followed by atomic-level structure refinement. The biological functions of the protein, including ligand-binding sites, enzyme commission number, and gene ontology terms, are then inferred from known protein function databases based on sequence and structure profile comparisons. I-TASSER is freely available as both an on-line server and a stand-alone package. This unit describes how to use the I-TASSER protocol to generate structure and function prediction and how to interpret the prediction results, as well as alternative approaches for further improving the I-TASSER modeling quality for distant-homologous and multi-domain protein targets. PMID:26678386

MultiSeq: unifying sequence and structure data for evolutionary analysis

PubMed Central

Roberts, Elijah; Eargle, John; Wright, Dan; Luthey-Schulten, Zaida

2006-01-01

Background Since the publication of the first draft of the human genome in 2000, bioinformatic data have been accumulating at an overwhelming pace. Currently, more than 3 million sequences and 35 thousand structures of proteins and nucleic acids are available in public databases. Finding correlations in and between these data to answer critical research questions is extremely challenging. This problem needs to be approached from several directions: information science to organize and search the data; information visualization to assist in recognizing correlations; mathematics to formulate statistical inferences; and biology to analyze chemical and physical properties in terms of sequence and structure changes. Results Here we present MultiSeq, a unified bioinformatics analysis environment that allows one to organize, display, align and analyze both sequence and structure data for proteins and nucleic acids. While special emphasis is placed on analyzing the data within the framework of evolutionary biology, the environment is also flexible enough to accommodate other usage patterns. The evolutionary approach is supported by the use of predefined metadata, adherence to standard ontological mappings, and the ability for the user to adjust these classifications using an electronic notebook. MultiSeq contains a new algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of a homologous group of distantly related proteins. The method, based on the multidimensional QR factorization of multiple sequence and structure alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. Conclusion MultiSeq is a major extension of the Multiple Alignment tool that is provided as part of VMD, a structural visualization program for analyzing molecular dynamics simulations. Both are freely distributed by the NIH Resource for Macromolecular Modeling and Bioinformatics and MultiSeq is included with VMD starting with version 1.8.5. The MultiSeq website has details on how to download and use the software: PMID:16914055

FunGene: the functional gene pipeline and repository.

PubMed

Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

2013-01-01

Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Protein mass analysis of histones.

PubMed

Galasinski, Scott C; Resing, Katheryn A; Ahn, Natalie G

2003-09-01

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.

Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation

NASA Astrophysics Data System (ADS)

Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.

2018-01-01

The analysis of intact proteins via mass spectrometry can offer several benefits to proteome characterization, although the majority of top-down experiments focus on proteoforms in a relatively low mass range (<30 kDa). Recent studies have focused on improving the analysis of larger intact proteins (up to 75 kDa), but they have also highlighted several challenges to be addressed. One major hurdle is the efficient dissociation of larger protein ions, which often to do not yield extensive fragmentation via conventional tandem MS methods. Here we describe the first application of activated ion electron transfer dissociation (AI-ETD) to proteins in the 30-70 kDa range. AI-ETD leverages infrared photo-activation concurrent to ETD reactions to improve sequence-informative product ion generation. This method generates more product ions and greater sequence coverage than conventional ETD, higher-energy collisional dissociation (HCD), and ETD combined with supplemental HCD activation (EThcD). Importantly, AI-ETD provides the most thorough protein characterization for every precursor ion charge state investigated in this study, making it suitable as a universal fragmentation method in top-down experiments. Additionally, we highlight several acquisition strategies that can benefit characterization of larger proteins with AI-ETD, including combination of spectra from multiple ETD reaction times for a given precursor ion, multiple spectral acquisitions of the same precursor ion, and combination of spectra from two different dissociation methods (e.g., AI-ETD and HCD). In all, AI-ETD shows great promise as a method for dissociating larger intact protein ions as top-down proteomics continues to advance into larger mass ranges. [Figure not available: see fulltext.

Benchmarking Inverse Statistical Approaches for Protein Structure and Design with Exactly Solvable Models.

PubMed

Jacquin, Hugo; Gilson, Amy; Shakhnovich, Eugene; Cocco, Simona; Monasson, Rémi

2016-05-01

Inverse statistical approaches to determine protein structure and function from Multiple Sequence Alignments (MSA) are emerging as powerful tools in computational biology. However the underlying assumptions of the relationship between the inferred effective Potts Hamiltonian and real protein structure and energetics remain untested so far. Here we use lattice protein model (LP) to benchmark those inverse statistical approaches. We build MSA of highly stable sequences in target LP structures, and infer the effective pairwise Potts Hamiltonians from those MSA. We find that inferred Potts Hamiltonians reproduce many important aspects of 'true' LP structures and energetics. Careful analysis reveals that effective pairwise couplings in inferred Potts Hamiltonians depend not only on the energetics of the native structure but also on competing folds; in particular, the coupling values reflect both positive design (stabilization of native conformation) and negative design (destabilization of competing folds). In addition to providing detailed structural information, the inferred Potts models used as protein Hamiltonian for design of new sequences are able to generate with high probability completely new sequences with the desired folds, which is not possible using independent-site models. Those are remarkable results as the effective LP Hamiltonians used to generate MSA are not simple pairwise models due to the competition between the folds. Our findings elucidate the reasons for the success of inverse approaches to the modelling of proteins from sequence data, and their limitations.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

PubMed

Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R

2005-09-01

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

PubMed Central

Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

1993-01-01

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

Predicting residue-wise contact orders in proteins by support vector regression.

PubMed

Song, Jiangning; Burrage, Kevin

2006-10-03

The residue-wise contact order (RWCO) describes the sequence separations between the residues of interest and its contacting residues in a protein sequence. It is a new kind of one-dimensional protein structure that represents the extent of long-range contacts and is considered as a generalization of contact order. Together with secondary structure, accessible surface area, the B factor, and contact number, RWCO provides comprehensive and indispensable important information to reconstructing the protein three-dimensional structure from a set of one-dimensional structural properties. Accurately predicting RWCO values could have many important applications in protein three-dimensional structure prediction and protein folding rate prediction, and give deep insights into protein sequence-structure relationships. We developed a novel approach to predict residue-wise contact order values in proteins based on support vector regression (SVR), starting from primary amino acid sequences. We explored seven different sequence encoding schemes to examine their effects on the prediction performance, including local sequence in the form of PSI-BLAST profiles, local sequence plus amino acid composition, local sequence plus molecular weight, local sequence plus secondary structure predicted by PSIPRED, local sequence plus molecular weight and amino acid composition, local sequence plus molecular weight and predicted secondary structure, and local sequence plus molecular weight, amino acid composition and predicted secondary structure. When using local sequences with multiple sequence alignments in the form of PSI-BLAST profiles, we could predict the RWCO distribution with a Pearson correlation coefficient (CC) between the predicted and observed RWCO values of 0.55, and root mean square error (RMSE) of 0.82, based on a well-defined dataset with 680 protein sequences. Moreover, by incorporating global features such as molecular weight and amino acid composition we could further improve the prediction performance with the CC to 0.57 and an RMSE of 0.79. In addition, combining the predicted secondary structure by PSIPRED was found to significantly improve the prediction performance and could yield the best prediction accuracy with a CC of 0.60 and RMSE of 0.78, which provided at least comparable performance compared with the other existing methods. The SVR method shows a prediction performance competitive with or at least comparable to the previously developed linear regression-based methods for predicting RWCO values. In contrast to support vector classification (SVC), SVR is very good at estimating the raw value profiles of the samples. The successful application of the SVR approach in this study reinforces the fact that support vector regression is a powerful tool in extracting the protein sequence-structure relationship and in estimating the protein structural profiles from amino acid sequences.

DLocalMotif: a discriminative approach for discovering local motifs in protein sequences.

PubMed

Mehdi, Ahmed M; Sehgal, Muhammad Shoaib B; Kobe, Bostjan; Bailey, Timothy L; Bodén, Mikael

2013-01-01

Local motifs are patterns of DNA or protein sequences that occur within a sequence interval relative to a biologically defined anchor or landmark. Current protein motif discovery methods do not adequately consider such constraints to identify biologically significant motifs that are only weakly over-represented but spatially confined. Using negatives, i.e. sequences known to not contain a local motif, can further increase the specificity of their discovery. This article introduces the method DLocalMotif that makes use of positional information and negative data for local motif discovery in protein sequences. DLocalMotif combines three scoring functions, measuring degrees of motif over-representation, entropy and spatial confinement, specifically designed to discriminatively exploit the availability of negative data. The method is shown to outperform current methods that use only a subset of these motif characteristics. We apply the method to several biological datasets. The analysis of peroxisomal targeting signals uncovers several novel motifs that occur immediately upstream of the dominant peroxisomal targeting signal-1 signal. The analysis of proline-tyrosine nuclear localization signals uncovers multiple novel motifs that overlap with C2H2 zinc finger domains. We also evaluate the method on classical nuclear localization signals and endoplasmic reticulum retention signals and find that DLocalMotif successfully recovers biologically relevant sequence properties. http://bioinf.scmb.uq.edu.au/dlocalmotif/

Sequence similarity is more relevant than species specificity in probabilistic backtranslation.

PubMed

Ferro, Alfredo; Giugno, Rosalba; Pigola, Giuseppe; Pulvirenti, Alfredo; Di Pietro, Cinzia; Purrello, Michele; Ragusa, Marco

2007-02-21

Backtranslation is the process of decoding a sequence of amino acids into the corresponding codons. All synthetic gene design systems include a backtranslation module. The degeneracy of the genetic code makes backtranslation potentially ambiguous since most amino acids are encoded by multiple codons. The common approach to overcome this difficulty is based on imitation of codon usage within the target species. This paper describes EasyBack, a new parameter-free, fully-automated software for backtranslation using Hidden Markov Models. EasyBack is not based on imitation of codon usage within the target species, but instead uses a sequence-similarity criterion. The model is trained with a set of proteins with known cDNA coding sequences, constructed from the input protein by querying the NCBI databases with BLAST. Unlike existing software, the proposed method allows the quality of prediction to be estimated. When tested on a group of proteins that show different degrees of sequence conservation, EasyBack outperforms other published methods in terms of precision. The prediction quality of a protein backtranslation methis markedly increased by replacing the criterion of most used codon in the same species with a Hidden Markov Model trained with a set of most similar sequences from all species. Moreover, the proposed method allows the quality of prediction to be estimated probabilistically.

Prediction of pi-turns in proteins using PSI-BLAST profiles and secondary structure information.

PubMed

Wang, Yan; Xue, Zhi-Dong; Shi, Xiao-Hong; Xu, Jin

2006-09-01

Due to the structural and functional importance of tight turns, some methods have been proposed to predict gamma-turns, beta-turns, and alpha-turns in proteins. In the past, studies of pi-turns were made, but not a single prediction approach has been developed so far. It will be useful to develop a method for identifying pi-turns in a protein sequence. In this paper, the support vector machine (SVM) method has been introduced to predict pi-turns from the amino acid sequence. The training and testing of this approach is performed with a newly collected data set of 640 non-homologous protein chains containing 1931 pi-turns. Different sequence encoding schemes have been explored in order to investigate their effects on the prediction performance. With multiple sequence alignment and predicted secondary structure, the final SVM model yields a Matthews correlation coefficient (MCC) of 0.556 by a 7-fold cross-validation. A web server implementing the prediction method is available at the following URL: http://210.42.106.80/piturn/.

A Survey of Protein Structures from Archaeal Viruses

PubMed Central

Dellas, Nikki; Lawrence, C. Martin; Young, Mark J.

2013-01-01

Viruses that infect the third domain of life, Archaea, are a newly emerging field of interest. To date, all characterized archaeal viruses infect archaea that thrive in extreme conditions, such as halophilic, hyperthermophilic, and methanogenic environments. Viruses in general, especially those replicating in extreme environments, contain highly mosaic genomes with open reading frames (ORFs) whose sequences are often dissimilar to all other known ORFs. It has been estimated that approximately 85% of virally encoded ORFs do not match known sequences in the nucleic acid databases, and this percentage is even higher for archaeal viruses (typically 90%–100%). This statistic suggests that either virus genomes represent a larger segment of sequence space and/or that viruses encode genes of novel fold and/or function. Because the overall three-dimensional fold of a protein evolves more slowly than its sequence, efforts have been geared toward structural characterization of proteins encoded by archaeal viruses in order to gain insight into their potential functions. In this short review, we provide multiple examples where structural characterization of archaeal viral proteins has indeed provided significant functional and evolutionary insight. PMID:25371334

Tutorial on Protein Ontology Resources

PubMed Central

Arighi, Cecilia; Drabkin, Harold; Christie, Karen R.; Ross, Karen; Natale, Darren

2017-01-01

The Protein Ontology (PRO) is the reference ontology for proteins in the Open Biomedical Ontologies (OBO) foundry and consists of three sub-ontologies representing protein classes of homologous genes, proteoforms (e.g., splice isoforms, sequence variants, and post-translationally modified forms), and protein complexes. PRO defines classes of proteins and protein complexes, both species-specific and species non-specific, and indicates their relationships in a hierarchical framework, supporting accurate protein annotation at the appropriate level of granularity, analyses of protein conservation across species, and semantic reasoning. In this first section of this chapter, we describe the PRO framework including categories of PRO terms and the relationship of PRO to other ontologies and protein resources. Next, we provide a tutorial about the PRO website (proconsortium.org) where users can browse and search the PRO hierarchy, view reports on individual PRO terms, and visualize relationships among PRO terms in a hierarchical table view, a multiple sequence alignment view, and a Cytoscape network view. Finally, we describe several examples illustrating the unique and rich information available in PRO. PMID:28150233

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

PubMed

Klaassen, V A; Boeshore, M; Dolja, V V; Falk, B W

1994-07-01

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

A coevolution analysis for identifying protein-protein interactions by Fourier transform.

PubMed

Yin, Changchuan; Yau, Stephen S-T

2017-01-01

Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI).

A coevolution analysis for identifying protein-protein interactions by Fourier transform

PubMed Central

Yin, Changchuan; Yau, Stephen S. -T.

2017-01-01

Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

Top-Down-Assisted Bottom-Up Method for Homologous Protein Sequencing: Hemoglobin from 33 Bird Species

NASA Astrophysics Data System (ADS)

Song, Yang; Laskay, Ünige A.; Vilcins, Inger-Marie E.; Barbour, Alan G.; Wysocki, Vicki H.

2015-11-01

Ticks are vectors for disease transmission because they are indiscriminant in their feeding on multiple vertebrate hosts, transmitting pathogens between their hosts. Identifying the hosts on which ticks have fed is important for disease prevention and intervention. We have previously shown that hemoglobin (Hb) remnants from a host on which a tick fed can be used to reveal the host's identity. For the present research, blood was collected from 33 bird species that are common in the U.S. as hosts for ticks but that have unknown Hb sequences. A top-down-assisted bottom-up mass spectrometry approach with a customized searching database, based on variability in known bird hemoglobin sequences, has been devised to facilitate fast and complete sequencing of hemoglobin from birds with unknown sequences. These hemoglobin sequences will be added to a hemoglobin database and used for tick host identification. The general approach has the potential to sequence any set of homologous proteins completely in a rapid manner.

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

PubMed

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

iASeq: integrative analysis of allele-specificity of protein-DNA interactions in multiple ChIP-seq datasets

PubMed Central

2012-01-01

Background ChIP-seq provides new opportunities to study allele-specific protein-DNA binding (ASB). However, detecting allelic imbalance from a single ChIP-seq dataset often has low statistical power since only sequence reads mapped to heterozygote SNPs are informative for discriminating two alleles. Results We develop a new method iASeq to address this issue by jointly analyzing multiple ChIP-seq datasets. iASeq uses a Bayesian hierarchical mixture model to learn correlation patterns of allele-specificity among multiple proteins. Using the discovered correlation patterns, the model allows one to borrow information across datasets to improve detection of allelic imbalance. Application of iASeq to 77 ChIP-seq samples from 40 ENCODE datasets and 1 genomic DNA sample in GM12878 cells reveals that allele-specificity of multiple proteins are highly correlated, and demonstrates the ability of iASeq to improve allelic inference compared to analyzing each individual dataset separately. Conclusions iASeq illustrates the value of integrating multiple datasets in the allele-specificity inference and offers a new tool to better analyze ASB. PMID:23194258

MODBASE, a database of annotated comparative protein structure models

PubMed Central

Pieper, Ursula; Eswar, Narayanan; Stuart, Ashley C.; Ilyin, Valentin A.; Sali, Andrej

2002-01-01

MODBASE (http://guitar.rockefeller.edu/modbase) is a relational database of annotated comparative protein structure models for all available protein sequences matched to at least one known protein structure. The models are calculated by MODPIPE, an automated modeling pipeline that relies on PSI-BLAST, IMPALA and MODELLER. MODBASE uses the MySQL relational database management system for flexible and efficient querying, and the MODVIEW Netscape plugin for viewing and manipulating multiple sequences and structures. It is updated regularly to reflect the growth of the protein sequence and structure databases, as well as improvements in the software for calculating the models. For ease of access, MODBASE is organized into different datasets. The largest dataset contains models for domains in 304 517 out of 539 171 unique protein sequences in the complete TrEMBL database (23 March 2001); only models based on significant alignments (PSI-BLAST E-value < 10–4) and models assessed to have the correct fold are included. Other datasets include models for target selection and structure-based annotation by the New York Structural Genomics Research Consortium, models for prediction of genes in the Drosophila melanogaster genome, models for structure determination of several ribosomal particles and models calculated by the MODWEB comparative modeling web server. PMID:11752309

Identification and Characterization of Novel Surface Proteins in Lactobacillus johnsonii and Lactobacillus gasseri

PubMed Central

Ventura, Marco; Jankovic, Ivana; Walker, D. Carey; Pridmore, R. David; Zink, Ralf

2002-01-01

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family. PMID:12450842

Sequence of a second gene encoding bovine submaxillary mucin: implication for mucin heterogeneity and cloning.

PubMed

Jiang, W; Woitach, J T; Gupta, D; Bhavanandan, V P

1998-10-20

Secreted epithelial mucins are extremely large and heterogeneous glycoproteins. We report the 5 kilobase DNA sequence of a second gene, BSM2, which encodes bovine submaxillary mucin. The determined nucleotide and deduced amino acid sequences of BSM2 are 95.2% and 92. 2% identical, respectively, to those of the previously described BSM1 gene isolated from the same cow. Further, the five predicted protein domains of the two genes are 100%, 94%, 93%, 77%, and 88% identical. Based on the above results, we propose that expression of multiple homologous core proteins from a single animal is a factor in generating diversity of saccharides in mucins and in providing resistance of the molecules to proteolysis. In addition, this work raises several important issues in mucin cloning such as assembling sequences from seemingly overlapping clones and deducing consensus sequences for nearly identical tandem repeats. Copyright 1998 Academic Press.

GWFASTA: server for FASTA search in eukaryotic and microbial genomes.

PubMed

Issac, Biju; Raghava, G P S

2002-09-01

Similarity searches are a powerful method for solving important biological problems such as database scanning, evolutionary studies, gene prediction, and protein structure prediction. FASTA is a widely used sequence comparison tool for rapid database scanning. Here we describe the GWFASTA server that was developed to assist the FASTA user in similarity searches against partially and/or completely sequenced genomes. GWFASTA consists of more than 60 microbial genomes, eight eukaryote genomes, and proteomes of annotatedgenomes. Infact, it provides the maximum number of databases for similarity searching from a single platform. GWFASTA allows the submission of more than one sequence as a single query for a FASTA search. It also provides integrated post-processing of FASTA output, including compositional analysis of proteins, multiple sequences alignment, and phylogenetic analysis. Furthermore, it summarizes the search results organism-wise for prokaryotes and chromosome-wise for eukaryotes. Thus, the integration of different tools for sequence analyses makes GWFASTA a powerful toolfor biologists.

Recent developments in the theory of protein folding: searching for the global energy minimum.

PubMed

Scheraga, H A

1996-04-16

Statistical mechanical theories and computer simulation are being used to gain an understanding of the fundamental features of protein folding. A major obstacle in the computation of protein structures is the multiple-minima problem arising from the existence of many local minima in the multidimensional energy landscape of the protein. This problem has been surmounted for small open-chain and cyclic peptides, and for regular-repeating sequences of models of fibrous proteins. Progress is being made in resolving this problem for globular proteins.

Sequence Diversity Diagram for comparative analysis of multiple sequence alignments.

PubMed

Sakai, Ryo; Aerts, Jan

2014-01-01

The sequence logo is a graphical representation of a set of aligned sequences, commonly used to depict conservation of amino acid or nucleotide sequences. Although it effectively communicates the amount of information present at every position, this visual representation falls short when the domain task is to compare between two or more sets of aligned sequences. We present a new visual presentation called a Sequence Diversity Diagram and validate our design choices with a case study. Our software was developed using the open-source program called Processing. It loads multiple sequence alignment FASTA files and a configuration file, which can be modified as needed to change the visualization. The redesigned figure improves on the visual comparison of two or more sets, and it additionally encodes information on sequential position conservation. In our case study of the adenylate kinase lid domain, the Sequence Diversity Diagram reveals unexpected patterns and new insights, for example the identification of subgroups within the protein subfamily. Our future work will integrate this visual encoding into interactive visualization tools to support higher level data exploration tasks.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

RNA regulatory networks diversified through curvature of the PUF protein scaffold

DOE PAGES

Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.; ...

2015-09-14

Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extendedmore » interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.« less

RNA regulatory networks diversified through curvature of the PUF protein scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P.

Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extendedmore » interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.« less

A minimalist model protein with multiple folding funnels

PubMed Central

Locker, C. Rebecca; Hernandez, Rigoberto

2001-01-01

Kinetic and structural studies of wild-type proteins such as prions and amyloidogenic proteins provide suggestive evidence that proteins may adopt multiple long-lived states in addition to the native state. All of these states differ structurally because they lie far apart in configuration space, but their stability is not necessarily caused by cooperative (nucleation) effects. In this study, a minimalist model protein is designed to exhibit multiple long-lived states to explore the dynamics of the corresponding wild-type proteins. The minimalist protein is modeled as a 27-monomer sequence confined to a cubic lattice with three different monomer types. An order parameter—the winding index—is introduced to characterize the extent of folding. The winding index has several advantages over other commonly used order parameters like the number of native contacts. It can distinguish between enantiomers, its calculation requires less computational time than the number of native contacts, and reduced-dimensional landscapes can be developed when the native state structure is not known a priori. The results for the designed model protein prove by existence that the rugged energy landscape picture of protein folding can be generalized to include protein “misfolding” into long-lived states. PMID:11470921

The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter

PubMed Central

Ohneda, Kinuko; Mirmira, Raghavendra G.; Wang, Juehu; Johnson, Jeffrey D.; German, Michael S.

2000-01-01

Activation of insulin gene transcription specifically in the pancreatic β cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-β cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins. PMID:10629047

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

PubMed Central

Vistein, Rachel; Puthenveedu, Manojkumar A.

2013-01-01

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling. PMID:24003153

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed Central

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-01-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery. Images PMID:8599930

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-02-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

RBT-GA: a novel metaheuristic for solving the Multiple Sequence Alignment problem.

PubMed

Taheri, Javid; Zomaya, Albert Y

2009-07-07

Multiple Sequence Alignment (MSA) has always been an active area of research in Bioinformatics. MSA is mainly focused on discovering biologically meaningful relationships among different sequences or proteins in order to investigate the underlying main characteristics/functions. This information is also used to generate phylogenetic trees. This paper presents a novel approach, namely RBT-GA, to solve the MSA problem using a hybrid solution methodology combining the Rubber Band Technique (RBT) and the Genetic Algorithm (GA) metaheuristic. RBT is inspired by the behavior of an elastic Rubber Band (RB) on a plate with several poles, which is analogues to locations in the input sequences that could potentially be biologically related. A GA attempts to mimic the evolutionary processes of life in order to locate optimal solutions in an often very complex landscape. RBT-GA is a population based optimization algorithm designed to find the optimal alignment for a set of input protein sequences. In this novel technique, each alignment answer is modeled as a chromosome consisting of several poles in the RBT framework. These poles resemble locations in the input sequences that are most likely to be correlated and/or biologically related. A GA-based optimization process improves these chromosomes gradually yielding a set of mostly optimal answers for the MSA problem. RBT-GA is tested with one of the well-known benchmarks suites (BALiBASE 2.0) in this area. The obtained results show that the superiority of the proposed technique even in the case of formidable sequences.

Initial genome sequencing and analysis of multiple myeloma

PubMed Central

Chapman, Michael A.; Lawrence, Michael S.; Keats, Jonathan J.; Cibulskis, Kristian; Sougnez, Carrie; Schinzel, Anna C.; Harview, Christina L.; Brunet, Jean-Philippe; Ahmann, Gregory J.; Adli, Mazhar; Anderson, Kenneth C.; Ardlie, Kristin G.; Auclair, Daniel; Baker, Angela; Bergsagel, P. Leif; Bernstein, Bradley E.; Drier, Yotam; Fonseca, Rafael; Gabriel, Stacey B.; Hofmeister, Craig C.; Jagannath, Sundar; Jakubowiak, Andrzej J.; Krishnan, Amrita; Levy, Joan; Liefeld, Ted; Lonial, Sagar; Mahan, Scott; Mfuko, Bunmi; Monti, Stefano; Perkins, Louise M.; Onofrio, Robb; Pugh, Trevor J.; Vincent Rajkumar, S.; Ramos, Alex H.; Siegel, David S.; Sivachenko, Andrey; Trudel, Suzanne; Vij, Ravi; Voet, Douglas; Winckler, Wendy; Zimmerman, Todd; Carpten, John; Trent, Jeff; Hahn, William C.; Garraway, Levi A.; Meyerson, Matthew; Lander, Eric S.; Getz, Gad; Golub, Todd R.

2013-01-01

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumor genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the dataset. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signaling was suggested by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge. PMID:21430775

ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

PubMed

Cheng, Yiming; Perocchi, Fabiana

2015-07-01

ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A new subfamily LIP of the major intrinsic proteins.

PubMed

Khabudaev, Kirill Vladimirovich; Petrova, Darya Petrovna; Grachev, Mikhail Aleksandrovich; Likhoshway, Yelena Valentinovna

2014-03-04

Proteins of the major intrinsic protein (MIP) family, or aquaporins, have been detected in almost all organisms. These proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules. We compared the predicted amino acid sequences of 20 MIPs from several algae species of the phylum Heterokontophyta (Kingdom Chromista) with the sequences of MIPs from other organisms. Multiple sequence alignments revealed motifs that were homologous to functionally important NPA motifs and the so-called ar/R-selective filter of glyceroporins and aquaporins. The MIP sequences of the studied chromists fell into several clusters that belonged to different groups of MIPs from a wide variety of organisms from different Kingdoms. Two of these proteins belong to Plasma membrane intrinsic proteins (PIPs), four of them belong to GlpF-like intrinsic proteins (GIPs), and one of them belongs to a specific MIPE subfamily from green algae. Three proteins belong to the unclassified MIPs, two of which are of bacterial origin. Eight of the studied MIPs contain an NPM-motif in place of the second conserved NPA-motif typical of the majority of MIPs. The MIPs of heterokonts within all detected clusters can differ from other MIPs in the same cluster regarding the structure of the ar/R-selective filter and other generally conserved motifs. We proposed placing nine MIPs from heterokonts into a new group, which we have named the LIPs (large intrinsic proteins). The possible substrate specificities of the studied MIPs are discussed.

Evolutionary profiles derived from the QR factorization of multiple structural alignments gives an economy of information.

PubMed

O'Donoghue, Patrick; Luthey-Schulten, Zaida

2005-02-25

We present a new algorithm, based on the multidimensional QR factorization, to remove redundancy from a multiple structural alignment by choosing representative protein structures that best preserve the phylogenetic tree topology of the homologous group. The classical QR factorization with pivoting, developed as a fast numerical solution to eigenvalue and linear least-squares problems of the form Ax=b, was designed to re-order the columns of A by increasing linear dependence. Removing the most linear dependent columns from A leads to the formation of a minimal basis set which well spans the phase space of the problem at hand. By recasting the problem of redundancy in multiple structural alignments into this framework, in which the matrix A now describes the multiple alignment, we adapted the QR factorization to produce a minimal basis set of protein structures which best spans the evolutionary (phase) space. The non-redundant and representative profiles obtained from this procedure, termed evolutionary profiles, are shown in initial results to outperform well-tested profiles in homology detection searches over a large sequence database. A measure of structural similarity between homologous proteins, Q(H), is presented. By properly accounting for the effect and presence of gaps, a phylogenetic tree computed using this metric is shown to be congruent with the maximum-likelihood sequence-based phylogeny. The results indicate that evolutionary information is indeed recoverable from the comparative analysis of protein structure alone. Applications of the QR ordering and this structural similarity metric to analyze the evolution of structure among key, universally distributed proteins involved in translation, and to the selection of representatives from an ensemble of NMR structures are also discussed.

[Exome sequencing revealed Allan-Herndon-Dudley syndrome underlying multiple disabilities].

PubMed

Arvio, Maria; Philips, Anju K; Ahvenainen, Minna; Somer, Mirja; Kalscheuer, Vera; Järvelä, Irma

2014-01-01

Normal function of the thyroid gland is the cornerstone of a child's mental development and physical growth. We describe a Finnish family, in which the diagnosis of three brothers became clear after investigations that lasted for more than 30 years. Two of the sons have already died. DNA analysis of the third one, a 16-year-old boy, revealed in exome sequencing of the complete X chromosome a mutation in the SLC16A2 gene, i.e. MCT8, coding for a thyroid hormone transport protein. Allan-Herndon-Dudley syndrome was thus shown to be the cause of multiple disabilities.

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

NASA Astrophysics Data System (ADS)

Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.

2017-09-01

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., 60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. [Figure not available: see fulltext.

Conserved interdomain linker promotes phase separation of the multivalent adaptor protein Nck

PubMed Central

Banjade, Sudeep; Wu, Qiong; Mittal, Anuradha; Peeples, William B.; Pappu, Rohit V.; Rosen, Michael K.

2015-01-01

The organization of membranes, the cytosol, and the nucleus of eukaryotic cells can be controlled through phase separation of lipids, proteins, and nucleic acids. Collective interactions of multivalent molecules mediated by modular binding domains can induce gelation and phase separation in several cytosolic and membrane-associated systems. The adaptor protein Nck has three SRC-homology 3 (SH3) domains that bind multiple proline-rich segments in the actin regulatory protein neuronal Wiskott-Aldrich syndrome protein (N-WASP) and an SH2 domain that binds to multiple phosphotyrosine sites in the adhesion protein nephrin, leading to phase separation. Here, we show that the 50-residue linker between the first two SH3 domains of Nck enhances phase separation of Nck/N-WASP/nephrin assemblies. Two linear motifs within this element, as well as its overall positively charged character, are important for this effect. The linker increases the driving force for self-assembly of Nck, likely through weak interactions with the second SH3 domain, and this effect appears to promote phase separation. The linker sequence is highly conserved, suggesting that the sequence determinants of the driving forces for phase separation may be generally important to Nck functions. Our studies demonstrate that linker regions between modular domains can contribute to the driving forces for self-assembly and phase separation of multivalent proteins. PMID:26553976

Sequence basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology

NASA Astrophysics Data System (ADS)

So, Christopher R.; Fears, Kenan P.; Leary, Dagmar H.; Scancella, Jenifer M.; Wang, Zheng; Liu, Jinny L.; Orihuela, Beatriz; Rittschof, Dan; Spillmann, Christopher M.; Wahl, Kathryn J.

2016-11-01

Barnacles adhere by producing a mixture of cement proteins (CPs) that organize into a permanently bonded layer displayed as nanoscale fibers. These cement proteins share no homology with any other marine adhesives, and a common sequence-basis that defines how nanostructures function as adhesives remains undiscovered. Here we demonstrate that a significant unidentified portion of acorn barnacle cement is comprised of low complexity proteins; they are organized into repetitive sequence blocks and found to maintain homology to silk motifs. Proteomic analysis of aggregate bands from PAGE gels reveal an abundance of Gly/Ala/Ser/Thr repeats exemplified by a prominent, previously unidentified, 43 kDa protein in the solubilized adhesive. Low complexity regions found throughout the cement proteome, as well as multiple lysyl oxidases and peroxidases, establish homology with silk-associated materials such as fibroin, silk gum sericin, and pyriform spidroins from spider silk. Distinct primary structures defined by homologous domains shed light on how barnacles use low complexity in nanofibers to enable adhesion, and serves as a starting point for unraveling the molecular architecture of a robust and unique class of adhesive nanostructures.

IBS: an illustrator for the presentation and visualization of biological sequences.

PubMed

Liu, Wenzhong; Xie, Yubin; Ma, Jiyong; Luo, Xiaotong; Nie, Peng; Zuo, Zhixiang; Lahrmann, Urs; Zhao, Qi; Zheng, Yueyuan; Zhao, Yong; Xue, Yu; Ren, Jian

2015-10-15

Biological sequence diagrams are fundamental for visualizing various functional elements in protein or nucleotide sequences that enable a summarization and presentation of existing information as well as means of intuitive new discoveries. Here, we present a software package called illustrator of biological sequences (IBS) that can be used for representing the organization of either protein or nucleotide sequences in a convenient, efficient and precise manner. Multiple options are provided in IBS, and biological sequences can be manipulated, recolored or rescaled in a user-defined mode. Also, the final representational artwork can be directly exported into a publication-quality figure. The standalone package of IBS was implemented in JAVA, while the online service was implemented in HTML5 and JavaScript. Both the standalone package and online service are freely available at http://ibs.biocuckoo.org. renjian.sysu@gmail.com or xueyu@hust.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

IBS: an illustrator for the presentation and visualization of biological sequences

PubMed Central

Liu, Wenzhong; Xie, Yubin; Ma, Jiyong; Luo, Xiaotong; Nie, Peng; Zuo, Zhixiang; Lahrmann, Urs; Zhao, Qi; Zheng, Yueyuan; Zhao, Yong; Xue, Yu; Ren, Jian

2015-01-01

Summary: Biological sequence diagrams are fundamental for visualizing various functional elements in protein or nucleotide sequences that enable a summarization and presentation of existing information as well as means of intuitive new discoveries. Here, we present a software package called illustrator of biological sequences (IBS) that can be used for representing the organization of either protein or nucleotide sequences in a convenient, efficient and precise manner. Multiple options are provided in IBS, and biological sequences can be manipulated, recolored or rescaled in a user-defined mode. Also, the final representational artwork can be directly exported into a publication-quality figure. Availability and implementation: The standalone package of IBS was implemented in JAVA, while the online service was implemented in HTML5 and JavaScript. Both the standalone package and online service are freely available at http://ibs.biocuckoo.org. Contact: renjian.sysu@gmail.com or xueyu@hust.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26069263

DendroBLAST: approximate phylogenetic trees in the absence of multiple sequence alignments.

PubMed

Kelly, Steven; Maini, Philip K

2013-01-01

The rapidly growing availability of genome information has created considerable demand for both fast and accurate phylogenetic inference algorithms. We present a novel method called DendroBLAST for reconstructing phylogenetic dendrograms/trees from protein sequences using BLAST. This method differs from other methods by incorporating a simple model of sequence evolution to test the effect of introducing sequence changes on the reliability of the bipartitions in the inferred tree. Using realistic simulated sequence data we demonstrate that this method produces phylogenetic trees that are more accurate than other commonly-used distance based methods though not as accurate as maximum likelihood methods from good quality multiple sequence alignments. In addition to tests on simulated data, we use DendroBLAST to generate input trees for a supertree reconstruction of the phylogeny of the Archaea. This independent analysis produces an approximate phylogeny of the Archaea that has both high precision and recall when compared to previously published analysis of the same dataset using conventional methods. Taken together these results demonstrate that approximate phylogenetic trees can be produced in the absence of multiple sequence alignments, and we propose that these trees will provide a platform for improving and informing downstream bioinformatic analysis. A web implementation of the DendroBLAST method is freely available for use at http://www.dendroblast.com/.

StralSV: assessment of sequence variability within similar 3D structures and application to polio RNA-dependent RNA polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zemla, A; Lang, D; Kostova, T

2010-11-29

Most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. A serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. Multiple sequence alignment methods are more satisfactory - still, they cannot provide reliable results at low levels of sequence identity. Our goal in the current work was to develop an algorithm that could overcome these difficulties and facilitatemore » the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. Here we present StralSV, a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. We apply StralSV in a study of the RNA-dependent RNA polymerase of poliovirus and demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique or that shared structural similarity with structures that are distantly related. By quantifying residue frequencies among many residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. We further demonstrate that considerable detailed structural and phylogenetic information can be derived from StralSV analyses. StralSV is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. StralSV analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position.« less

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

PubMed Central

Marck, C

1988-01-01

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds. PMID:2832831

RNA regulators responding to ribosomal protein S15 are frequent in sequence space

PubMed Central

Slinger, Betty L.; Meyer, Michelle M.

2016-01-01

There are several natural examples of distinct RNA structures that interact with the same ligand to regulate the expression of homologous genes in different organisms. One essential question regarding this phenomenon is whether such RNA regulators are the result of convergent or divergent evolution. Are the RNAs derived from some common ancestor and diverged to the point where we cannot identify the similarity, or have multiple solutions to the same biological problem arisen independently? A key variable in assessing these alternatives is how frequently such regulators arise within sequence space. Ribosomal protein S15 is autogenously regulated via an RNA regulator in many bacterial species; four apparently distinct regulators have been functionally validated in different bacterial phyla. Here, we explore how frequently such regulators arise within a partially randomized sequence population. We find many RNAs that interact specifically with ribosomal protein S15 from Geobacillus kaustophilus with biologically relevant dissociation constants. Furthermore, of the six sequences we characterize, four show regulatory activity in an Escherichia coli reporter assay. Subsequent footprinting and mutagenesis analysis indicates that protein binding proximal to regulatory features such as the Shine–Dalgarno sequence is sufficient to enable regulation, suggesting that regulation in response to S15 is relatively easily acquired. PMID:27580716

A new fusion protein platform for quantitatively measuring activity of multiple proteases

PubMed Central

2014-01-01

Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity. PMID:24649897

Abnormality in catalase import into peroxisomes leads to severe neurological disorder

PubMed Central

Sheikh, Faruk G.; Pahan, Kalipada; Khan, Mushfiquddin; Barbosa, Ernest; Singh, Inderjit

1998-01-01

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease. PMID:9501198

Evidence-based green algal genomics reveals marine diversity and ancestral characteristics of land plants.

PubMed

van Baren, Marijke J; Bachy, Charles; Reistetter, Emily Nahas; Purvine, Samuel O; Grimwood, Jane; Sudek, Sebastian; Yu, Hang; Poirier, Camille; Deerinck, Thomas J; Kuo, Alan; Grigoriev, Igor V; Wong, Chee-Hong; Smith, Richard D; Callister, Stephen J; Wei, Chia-Lin; Schmutz, Jeremy; Worden, Alexandra Z

2016-03-31

Prasinophytes are widespread marine green algae that are related to plants. Cellular abundance of the prasinophyte Micromonas has reportedly increased in the Arctic due to climate-induced changes. Thus, studies of these unicellular eukaryotes are important for marine ecology and for understanding Viridiplantae evolution and diversification. We generated evidence-based Micromonas gene models using proteomics and RNA-Seq to improve prasinophyte genomic resources. First, sequences of four chromosomes in the 22 Mb Micromonas pusilla (CCMP1545) genome were finished. Comparison with the finished 21 Mb genome of Micromonas commoda (RCC299; named herein) shows they share ≤8,141 of ~10,000 protein-encoding genes, depending on the analysis method. Unlike RCC299 and other sequenced eukaryotes, CCMP1545 has two abundant repetitive intron types and a high percent (26 %) GC splice donors. Micromonas has more genus-specific protein families (19 %) than other genome sequenced prasinophytes (11 %). Comparative analyses using predicted proteomes from other prasinophytes reveal proteins likely related to scale formation and ancestral photosynthesis. Our studies also indicate that peptidoglycan (PG) biosynthesis enzymes have been lost in multiple independent events in select prasinophytes and plants. However, CCMP1545, polar Micromonas CCMP2099 and prasinophytes from other classes retain the entire PG pathway, like moss and glaucophyte algae. Surprisingly, multiple vascular plants also have the PG pathway, except the Penicillin-Binding Protein, and share a unique bi-domain protein potentially associated with the pathway. Alongside Micromonas experiments using antibiotics that halt bacterial PG biosynthesis, the findings highlight unrecognized phylogenetic complexity in PG-pathway retention and implicate a role in chloroplast structure or division in several extant Viridiplantae lineages. Extensive differences in gene loss and architecture between related prasinophytes underscore their divergence. PG biosynthesis genes from the cyanobacterial endosymbiont that became the plastid, have been selectively retained in multiple plants and algae, implying a biological function. Our studies provide robust genomic resources for emerging model algae, advancing knowledge of marine phytoplankton and plant evolution.

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

PubMed

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Egg Case Silk Gene Sequences from Argiope Spiders: Evidence for Multiple Loci and a Loss of Function Between Paralogs

PubMed Central

Chaw, R. Crystal; Collin, Matthew; Wimmer, Marjorie; Helmrick, Kara-Leigh; Hayashi, Cheryl Y.

2017-01-01

Spiders swath their eggs with silk to protect developing embryos and hatchlings. Egg case silks, like other fibrous spider silks, are primarily composed of proteins called spidroins (spidroin = spider-fibroin). Silks, and thus spidroins, are important throughout the lives of spiders, yet the evolution of spidroin genes has been relatively understudied. Spidroin genes are notoriously difficult to sequence because they are typically very long (≥ 10 kb of coding sequence) and highly repetitive. Here, we investigate the evolution of spider silk genes through long-read sequencing of Bacterial Artificial Chromosome (BAC) clones. We demonstrate that the silver garden spider Argiope argentata has multiple egg case spidroin loci with a loss of function at one locus. We also use degenerate PCR primers to search the genomic DNA of congeneric species and find evidence for multiple egg case spidroin loci in other Argiope spiders. Comparative analyses show that these multiple loci are more similar at the nucleotide level within a species than between species. This pattern is consistent with concerted evolution homogenizing gene copies within a genome. More complicated explanations include convergent evolution or recent independent gene duplications within each species. PMID:29127108

Disorder and function: a review of the dehydrin protein family

PubMed Central

Graether, Steffen P.; Boddington, Kelly F.

2014-01-01

Dehydration proteins (dehydrins) are group 2 members of the late embryogenesis abundant (LEA) protein family. The protein architecture of dehydrins can be described by the presence of three types of conserved sequence motifs that have been named the K-, Y-, and S-segments. By definition, a dehydrin must contain at least one copy of the lysine-rich K-segment. Abiotic stresses such as drought, cold, and salinity cause the upregulation of dehydrin mRNA and protein levels. Despite the large body of genetic and protein evidence of the importance of these proteins in stress response, the in vivo protective mechanism is not fully known. In vitro experimental evidence from biochemical assays and localization experiments suggests multiple roles for dehydrins, including membrane protection, cryoprotection of enzymes, and protection from reactive oxygen species. Membrane binding by dehydrins is likely to be as a peripheral membrane protein, since the protein sequences are highly hydrophilic and contain many charged amino acids. Because of this, dehydrins in solution are intrinsically disordered proteins, that is, they have no well-defined secondary or tertiary structure. Despite their disorder, dehydrins have been shown to gain structure when bound to ligands such as membranes, and to possibly change their oligomeric state when bound to ions. We review what is currently known about dehydrin sequences and their structures, and examine the various ligands that have been shown to bind to this family of proteins. PMID:25400646

Recombinant Collagenlike Proteins

NASA Technical Reports Server (NTRS)

Fertala, Andzej

2007-01-01

A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

Targeted Quantitation of Proteins by Mass Spectrometry

PubMed Central

2013-01-01

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

Targeted quantitation of proteins by mass spectrometry.

PubMed

Liebler, Daniel C; Zimmerman, Lisa J

2013-06-04

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement.

Protein-Level Integration Strategy of Multiengine MS Spectra Search Results for Higher Confidence and Sequence Coverage.

PubMed

Zhao, Panpan; Zhong, Jiayong; Liu, Wanting; Zhao, Jing; Zhang, Gong

2017-12-01

Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20. As expected, the union of seven engines resulted in a boosted false identification, whereas the intersection of seven engines remarkably decreased the identification power. We found that identifications of at least two out of seven engines resulted in maximizing the protein identification power while minimizing the ratio of suspicious/translation-supported identifications (STR), as monitored by our STR index, based on RNC-Seq. Furthermore, this strategy also significantly improves the peptides coverage of the protein amino acid sequence. In summary, we demonstrated a simple strategy to significantly improve the performance for shotgun mass spectrometry by protein-level integrating multiple search engines, maximizing the utilization of the current MS spectra without additional experimental work.

Prediction of rat protein subcellular localization with pseudo amino acid composition based on multiple sequential features.

PubMed

Shi, Ruijia; Xu, Cunshuan

2011-06-01

The study of rat proteins is an indispensable task in experimental medicine and drug development. The function of a rat protein is closely related to its subcellular location. Based on the above concept, we construct the benchmark rat proteins dataset and develop a combined approach for predicting the subcellular localization of rat proteins. From protein primary sequence, the multiple sequential features are obtained by using of discrete Fourier analysis, position conservation scoring function and increment of diversity, and these sequential features are selected as input parameters of the support vector machine. By the jackknife test, the overall success rate of prediction is 95.6% on the rat proteins dataset. Our method are performed on the apoptosis proteins dataset and the Gram-negative bacterial proteins dataset with the jackknife test, the overall success rates are 89.9% and 96.4%, respectively. The above results indicate that our proposed method is quite promising and may play a complementary role to the existing predictors in this area.

A cohort of new adhesive proteins identified from transcriptomic analysis of mussel foot glands.

PubMed

DeMartini, Daniel G; Errico, John M; Sjoestroem, Sebastian; Fenster, April; Waite, J Herbert

2017-06-01

The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion. © 2017 The Author(s).

Prediction of protein long-range contacts using an ensemble of genetic algorithm classifiers with sequence profile centers.

PubMed

Chen, Peng; Li, Jinyan

2010-05-17

Prediction of long-range inter-residue contacts is an important topic in bioinformatics research. It is helpful for determining protein structures, understanding protein foldings, and therefore advancing the annotation of protein functions. In this paper, we propose a novel ensemble of genetic algorithm classifiers (GaCs) to address the long-range contact prediction problem. Our method is based on the key idea called sequence profile centers (SPCs). Each SPC is the average sequence profiles of residue pairs belonging to the same contact class or non-contact class. GaCs train on multiple but different pairs of long-range contact data (positive data) and long-range non-contact data (negative data). The negative data sets, having roughly the same sizes as the positive ones, are constructed by random sampling over the original imbalanced negative data. As a result, about 21.5% long-range contacts are correctly predicted. We also found that the ensemble of GaCs indeed makes an accuracy improvement by around 5.6% over the single GaC. Classifiers with the use of sequence profile centers may advance the long-range contact prediction. In line with this approach, key structural features in proteins would be determined with high efficiency and accuracy.

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

PubMed Central

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-01-01

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Introduction to bioinformatics.

PubMed

Can, Tolga

2014-01-01

Bioinformatics is an interdisciplinary field mainly involving molecular biology and genetics, computer science, mathematics, and statistics. Data intensive, large-scale biological problems are addressed from a computational point of view. The most common problems are modeling biological processes at the molecular level and making inferences from collected data. A bioinformatics solution usually involves the following steps: Collect statistics from biological data. Build a computational model. Solve a computational modeling problem. Test and evaluate a computational algorithm. This chapter gives a brief introduction to bioinformatics by first providing an introduction to biological terminology and then discussing some classical bioinformatics problems organized by the types of data sources. Sequence analysis is the analysis of DNA and protein sequences for clues regarding function and includes subproblems such as identification of homologs, multiple sequence alignment, searching sequence patterns, and evolutionary analyses. Protein structures are three-dimensional data and the associated problems are structure prediction (secondary and tertiary), analysis of protein structures for clues regarding function, and structural alignment. Gene expression data is usually represented as matrices and analysis of microarray data mostly involves statistics analysis, classification, and clustering approaches. Biological networks such as gene regulatory networks, metabolic pathways, and protein-protein interaction networks are usually modeled as graphs and graph theoretic approaches are used to solve associated problems such as construction and analysis of large-scale networks.

NLSdb-major update for database of nuclear localization signals and nuclear export signals.

PubMed

Bernhofer, Michael; Goldberg, Tatyana; Wolf, Silvana; Ahmed, Mohamed; Zaugg, Julian; Boden, Mikael; Rost, Burkhard

2018-01-04

NLSdb is a database collecting nuclear export signals (NES) and nuclear localization signals (NLS) along with experimentally annotated nuclear and non-nuclear proteins. NES and NLS are short sequence motifs related to protein transport out of and into the nucleus. The updated NLSdb now contains 2253 NLS and introduces 398 NES. The potential sets of novel NES and NLS have been generated by a simple 'in silico mutagenesis' protocol. We started with motifs annotated by experiments. In step 1, we increased specificity such that no known non-nuclear protein matched the refined motif. In step 2, we increased the sensitivity trying to match several different families with a motif. We then iterated over steps 1 and 2. The final set of 2253 NLS motifs matched 35% of 8421 experimentally verified nuclear proteins (up from 21% for the previous version) and none of 18 278 non-nuclear proteins. We updated the web interface providing multiple options to search protein sequences for NES and NLS motifs, and to evaluate your own signal sequences. NLSdb can be accessed via Rostlab services at: https://rostlab.org/services/nlsdb/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of a Conserved Non-Protein-Coding Genomic Element that Plays an Essential Role in Alphabaculovirus Pathogenesis

PubMed Central

Kikhno, Irina

2014-01-01

Highly homologous sequences 154–157 bp in length grouped under the name of “conserved non-protein-coding element” (CNE) were revealed in all of the sequenced genomes of baculoviruses belonging to the genus Alphabaculovirus. A CNE alignment led to the detection of a set of highly conserved nucleotide clusters that occupy strictly conserved positions in the CNE sequence. The significant length of the CNE and conservation of both its length and cluster architecture were identified as a combination of characteristics that make this CNE different from known viral non-coding functional sequences. The essential role of the CNE in the Alphabaculovirus life cycle was demonstrated through the use of a CNE-knockout Autographa californica multiple nucleopolyhedrovirus (AcMNPV) bacmid. It was shown that the essential function of the CNE was not mediated by the presumed expression activities of the protein- and non-protein-coding genes that overlap the AcMNPV CNE. On the basis of the presented data, the AcMNPV CNE was categorized as a complex-structured, polyfunctional genomic element involved in an essential DNA transaction that is associated with an undefined function of the baculovirus genome. PMID:24740153

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination.

PubMed

Kanduc, Darja; Fasano, Candida; Capone, Giovanni; Pesce Delfino, Antonella; Calabrò, Michele; Polimeno, Lorenzo

2015-01-01

Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination.

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

PubMed Central

Drobni, Mirva; Hallberg, Kristina; Öhman, Ulla; Birve, Anna; Persson, Karina; Johansson, Ingegerd; Strömberg, Nicklas

2006-01-01

Background Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. Results Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galβ-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. Conclusion The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated. PMID:16686953

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th-century pandemics.

PubMed

Pasricha, Gunisha; Mishra, Akhilesh C; Chakrabarti, Alok K

2013-07-01

PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Analysis showed that 96·4% of the H5N1 influenza viruses harbored full-length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th-century pandemic influenza viruses contained full-length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human- and avian host-specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host-specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity. © 2012 John Wiley & Sons Ltd.

A discriminative method for family-based protein remote homology detection that combines inductive logic programming and propositional models

PubMed Central

2011-01-01

Background Remote homology detection is a hard computational problem. Most approaches have trained computational models by using either full protein sequences or multiple sequence alignments (MSA), including all positions. However, when we deal with proteins in the "twilight zone" we can observe that only some segments of sequences (motifs) are conserved. We introduce a novel logical representation that allows us to represent physico-chemical properties of sequences, conserved amino acid positions and conserved physico-chemical positions in the MSA. From this, Inductive Logic Programming (ILP) finds the most frequent patterns (motifs) and uses them to train propositional models, such as decision trees and support vector machines (SVM). Results We use the SCOP database to perform our experiments by evaluating protein recognition within the same superfamily. Our results show that our methodology when using SVM performs significantly better than some of the state of the art methods, and comparable to other. However, our method provides a comprehensible set of logical rules that can help to understand what determines a protein function. Conclusions The strategy of selecting only the most frequent patterns is effective for the remote homology detection. This is possible through a suitable first-order logical representation of homologous properties, and through a set of frequent patterns, found by an ILP system, that summarizes essential features of protein functions. PMID:21429187

A discriminative method for family-based protein remote homology detection that combines inductive logic programming and propositional models.

PubMed

Bernardes, Juliana S; Carbone, Alessandra; Zaverucha, Gerson

2011-03-23

Remote homology detection is a hard computational problem. Most approaches have trained computational models by using either full protein sequences or multiple sequence alignments (MSA), including all positions. However, when we deal with proteins in the "twilight zone" we can observe that only some segments of sequences (motifs) are conserved. We introduce a novel logical representation that allows us to represent physico-chemical properties of sequences, conserved amino acid positions and conserved physico-chemical positions in the MSA. From this, Inductive Logic Programming (ILP) finds the most frequent patterns (motifs) and uses them to train propositional models, such as decision trees and support vector machines (SVM). We use the SCOP database to perform our experiments by evaluating protein recognition within the same superfamily. Our results show that our methodology when using SVM performs significantly better than some of the state of the art methods, and comparable to other. However, our method provides a comprehensible set of logical rules that can help to understand what determines a protein function. The strategy of selecting only the most frequent patterns is effective for the remote homology detection. This is possible through a suitable first-order logical representation of homologous properties, and through a set of frequent patterns, found by an ILP system, that summarizes essential features of protein functions.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

Finding the target sites of RNA-binding proteins

PubMed Central

Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

2014-01-01

RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

Evolution of the vertebrate insulin receptor substrate (Irs) gene family.

PubMed

Al-Salam, Ahmad; Irwin, David M

2017-06-23

Insulin receptor substrate (Irs) proteins are essential for insulin signaling as they allow downstream effectors to dock with, and be activated by, the insulin receptor. A family of four Irs proteins have been identified in mice, however the gene for one of these, IRS3, has been pseudogenized in humans. While it is known that the Irs gene family originated in vertebrates, it is not known when it originated and which members are most closely related to each other. A better understanding of the evolution of Irs genes and proteins should provide insight into the regulation of metabolism by insulin. Multiple genes for Irs proteins were identified in a wide variety of vertebrate species. Phylogenetic and genomic neighborhood analyses indicate that this gene family originated very early in vertebrae evolution. Most Irs genes were duplicated and retained in fish after the fish-specific genome duplication. Irs genes have been lost of various lineages, including Irs3 in primates and birds and Irs1 in most fish. Irs3 and Irs4 experienced an episode of more rapid protein sequence evolution on the ancestral mammalian lineage. Comparisons of the conservation of the proteins sequences among Irs paralogs show that domains involved in binding to the plasma membrane and insulin receptors are most strongly conserved, while divergence has occurred in sequences involved in interacting with downstream effector proteins. The Irs gene family originated very early in vertebrate evolution, likely through genome duplications, and in parallel with duplications of other components of the insulin signaling pathway, including insulin and the insulin receptor. While the N-terminal sequences of these proteins are conserved among the paralogs, changes in the C-terminal sequences likely allowed changes in biological function.

Experimental Aspects of Polarization Optimized Experiments (POE) for Magic Angle Spinning Solid-State NMR of Microcrystalline and Membrane-Bound Proteins.

PubMed

Gopinath, T; Veglia, Gianluigi

2018-01-01

Conventional NMR pulse sequences record one spectrum per experiment, while spending most of the time waiting for the spin system to return to the equilibrium. As a result, a full set of multidimensional NMR experiments for biological macromolecules may take up to several months to complete. Here, we present a practical guide for setting up a new class of MAS solid-state NMR experiments (POE or polarization optimized experiments) that enable the simultaneous acquisition of multiple spectra of proteins, accelerating data acquisition. POE exploit the long-lived 15 N polarization of isotopically labeled proteins and enable one to obtain up to eight spectra, by concatenating classical NMR pulse sequences. This new strategy propels data throughput of solid-state NMR spectroscopy of fibers, microcrystalline preparations, as well as membrane proteins.

MUSCLE: multiple sequence alignment with high accuracy and high throughput.

PubMed

Edgar, Robert C

2004-01-01

We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

Structural phylogeny by profile extraction and multiple superimposition using electrostatic congruence as a discriminator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Sandeep; Rao, Basuthkar J.; Baker, Nathan A.

2013-04-01

Phylogenetic analysis of proteins using multiple sequence alignment (MSA) assumes an underlying evolutionary relationship in these proteins which occasionally remains undetected due to considerable sequence divergence. Structural alignment programs have been developed to unravel such fuzzy relationships. However, none of these structure based methods have used electrostatic properties to discriminate between spatially equivalent residues. We present a methodology for MSA of a set of related proteins with known structures using electrostatic properties as an additional discriminator (STEEP). STEEP first extracts a profile, then generates a multiple structural superimposition providing a consolidated spatial framework for comparing residues and finally emits themore » MSA. Residues that are aligned differently by including or excluding electrostatic properties can be targeted by directed evolution experiments to transform the enzymatic properties of one protein into another. We have compared STEEP results to those obtained from a MSA program (ClustalW) and a structural alignment method (MUSTANG) for chymotrypsin serine proteases. Subsequently, we used PhyML to generate phylogenetic trees for the serine and metallo-β-lactamase superfamilies from the STEEP generated MSA, and corroborated the accepted relationships in these superfamilies. We have observed that STEEP acts as a functional classifier when electrostatic congruence is used as a discriminator, and thus identifies potential targets for directed evolution experiments. In summary, STEEP is unique among phylogenetic methods for its ability to use electrostatic congruence to specify mutations that might be the source of the functional divergence in a protein family. Based on our results, we also hypothesize that the active site and its close vicinity contains enough information to infer the correct phylogeny for related proteins.« less

RBT-GA: a novel metaheuristic for solving the multiple sequence alignment problem

PubMed Central

Taheri, Javid; Zomaya, Albert Y

2009-01-01

Background Multiple Sequence Alignment (MSA) has always been an active area of research in Bioinformatics. MSA is mainly focused on discovering biologically meaningful relationships among different sequences or proteins in order to investigate the underlying main characteristics/functions. This information is also used to generate phylogenetic trees. Results This paper presents a novel approach, namely RBT-GA, to solve the MSA problem using a hybrid solution methodology combining the Rubber Band Technique (RBT) and the Genetic Algorithm (GA) metaheuristic. RBT is inspired by the behavior of an elastic Rubber Band (RB) on a plate with several poles, which is analogues to locations in the input sequences that could potentially be biologically related. A GA attempts to mimic the evolutionary processes of life in order to locate optimal solutions in an often very complex landscape. RBT-GA is a population based optimization algorithm designed to find the optimal alignment for a set of input protein sequences. In this novel technique, each alignment answer is modeled as a chromosome consisting of several poles in the RBT framework. These poles resemble locations in the input sequences that are most likely to be correlated and/or biologically related. A GA-based optimization process improves these chromosomes gradually yielding a set of mostly optimal answers for the MSA problem. Conclusion RBT-GA is tested with one of the well-known benchmarks suites (BALiBASE 2.0) in this area. The obtained results show that the superiority of the proposed technique even in the case of formidable sequences. PMID:19594869

Quaranfil, Johnston Atoll, and Lake Chad viruses are novel members of the family Orthomyxoviridae.

PubMed

Presti, Rachel M; Zhao, Guoyan; Beatty, Wandy L; Mihindukulasuriya, Kathie A; da Rosa, Amelia P A Travassos; Popov, Vsevolod L; Tesh, Robert B; Virgin, Herbert W; Wang, David

2009-11-01

Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae.

Quaranfil, Johnston Atoll, and Lake Chad Viruses Are Novel Members of the Family Orthomyxoviridae▿

PubMed Central

Presti, Rachel M.; Zhao, Guoyan; Beatty, Wandy L.; Mihindukulasuriya, Kathie A.; Travassos da Rosa, Amelia P. A.; Popov, Vsevolod L.; Tesh, Robert B.; Virgin, Herbert W.; Wang, David

2009-01-01

Arboviral infections are an important cause of emerging infections due to the movements of humans, animals, and hematophagous arthropods. Quaranfil virus (QRFV) is an unclassified arbovirus originally isolated from children with mild febrile illness in Quaranfil, Egypt, in 1953. It has subsequently been isolated in multiple geographic areas from ticks and birds. We used high-throughput sequencing to classify QRFV as a novel orthomyxovirus. The genome of this virus is comprised of multiple RNA segments; five were completely sequenced. Proteins with limited amino acid similarity to conserved domains in polymerase (PA, PB1, and PB2) and hemagglutinin (HA) genes from known orthomyxoviruses were predicted to be present in four of the segments. The fifth sequenced segment shared no detectable similarity to any protein and is of uncertain function. The end-terminal sequences of QRFV are conserved between segments and are different from those of the known orthomyxovirus genera. QRFV is known to cross-react serologically with two other unclassified viruses, Johnston Atoll virus (JAV) and Lake Chad virus (LKCV). The complete open reading frames of PB1 and HA were sequenced for JAV, while a fragment of PB1 of LKCV was identified by mass sequencing. QRFV and JAV PB1 and HA shared 80% and 70% amino acid identity to each other, respectively; the LKCV PB1 fragment shared 83% amino acid identity with the corresponding region of QRFV PB1. Based on phylogenetic analyses, virion ultrastructural features, and the unique end-terminal sequences identified, we propose that QRFV, JAV, and LKCV comprise a novel genus of the family Orthomyxoviridae. PMID:19726499

Computational Identification and Functional Predictions of Long Noncoding RNA in Zea mays

PubMed Central

Boerner, Susan; McGinnis, Karen M.

2012-01-01

Background Computational analysis of cDNA sequences from multiple organisms suggests that a large portion of transcribed DNA does not code for a functional protein. In mammals, noncoding transcription is abundant, and often results in functional RNA molecules that do not appear to encode proteins. Many long noncoding RNAs (lncRNAs) appear to have epigenetic regulatory function in humans, including HOTAIR and XIST. While epigenetic gene regulation is clearly an essential mechanism in plants, relatively little is known about the presence or function of lncRNAs in plants. Methodology/Principal Findings To explore the connection between lncRNA and epigenetic regulation of gene expression in plants, a computational pipeline using the programming language Python has been developed and applied to maize full length cDNA sequences to identify, classify, and localize potential lncRNAs. The pipeline was used in parallel with an SVM tool for identifying ncRNAs to identify the maximal number of ncRNAs in the dataset. Although the available library of sequences was small and potentially biased toward protein coding transcripts, 15% of the sequences were predicted to be noncoding. Approximately 60% of these sequences appear to act as precursors for small RNA molecules and may function to regulate gene expression via a small RNA dependent mechanism. ncRNAs were predicted to originate from both genic and intergenic loci. Of the lncRNAs that originated from genic loci, ∼20% were antisense to the host gene loci. Conclusions/Significance Consistent with similar studies in other organisms, noncoding transcription appears to be widespread in the maize genome. Computational predictions indicate that maize lncRNAs may function to regulate expression of other genes through multiple RNA mediated mechanisms. PMID:22916204

Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.

PubMed Central

del Angel, R M; Papavassiliou, A G; Fernández-Tomás, C; Silverstein, S J; Racaniello, V R

1989-01-01

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody. Images PMID:2554308

Evolution of bacterial-like phosphoprotein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer.

PubMed

Uhrig, R Glen; Kerk, David; Moorhead, Greg B

2013-12-01

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

MUFOLD-SS: New deep inception-inside-inception networks for protein secondary structure prediction.

PubMed

Fang, Chao; Shang, Yi; Xu, Dong

2018-05-01

Protein secondary structure prediction can provide important information for protein 3D structure prediction and protein functions. Deep learning offers a new opportunity to significantly improve prediction accuracy. In this article, a new deep neural network architecture, named the Deep inception-inside-inception (Deep3I) network, is proposed for protein secondary structure prediction and implemented as a software tool MUFOLD-SS. The input to MUFOLD-SS is a carefully designed feature matrix corresponding to the primary amino acid sequence of a protein, which consists of a rich set of information derived from individual amino acid, as well as the context of the protein sequence. Specifically, the feature matrix is a composition of physio-chemical properties of amino acids, PSI-BLAST profile, and HHBlits profile. MUFOLD-SS is composed of a sequence of nested inception modules and maps the input matrix to either eight states or three states of secondary structures. The architecture of MUFOLD-SS enables effective processing of local and global interactions between amino acids in making accurate prediction. In extensive experiments on multiple datasets, MUFOLD-SS outperformed the best existing methods and other deep neural networks significantly. MUFold-SS can be downloaded from http://dslsrv8.cs.missouri.edu/~cf797/MUFoldSS/download.html. © 2018 Wiley Periodicals, Inc.

Network-based function prediction and interactomics: the case for metabolic enzymes.

PubMed

Janga, S C; Díaz-Mejía, J Javier; Moreno-Hagelsieb, G

2011-01-01

As sequencing technologies increase in power, determining the functions of unknown proteins encoded by the DNA sequences so produced becomes a major challenge. Functional annotation is commonly done on the basis of amino-acid sequence similarity alone. Long after sequence similarity becomes undetectable by pair-wise comparison, profile-based identification of homologs can often succeed due to the conservation of position-specific patterns, important for a protein's three dimensional folding and function. Nevertheless, prediction of protein function from homology-driven approaches is not without problems. Homologous proteins might evolve different functions and the power of homology detection has already started to reach its maximum. Computational methods for inferring protein function, which exploit the context of a protein in cellular networks, have come to be built on top of homology-based approaches. These network-based functional inference techniques provide both a first hand hint into a proteins' functional role and offer complementary insights to traditional methods for understanding the function of uncharacterized proteins. Most recent network-based approaches aim to integrate diverse kinds of functional interactions to boost both coverage and confidence level. These techniques not only promise to solve the moonlighting aspect of proteins by annotating proteins with multiple functions, but also increase our understanding on the interplay between different functional classes in a cell. In this article we review the state of the art in network-based function prediction and describe some of the underlying difficulties and successes. Given the volume of high-throughput data that is being reported the time is ripe to employ these network-based approaches, which can be used to unravel the functions of the uncharacterized proteins accumulating in the genomic databases. © 2010 Elsevier Inc. All rights reserved.

Application of an E. coli signal sequence as a versatile inclusion body tag.

PubMed

Jong, Wouter S P; Vikström, David; Houben, Diane; van den Berg van Saparoea, H Bart; de Gier, Jan-Willem; Luirink, Joen

2017-03-21

Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence

PubMed Central

Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

2016-01-01

Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain duplications, the reconstruction of the history of protein architectures, and the estimation of protein domain age. Website and software: http://www.lcqb.upmc.fr/CLADE. PMID:27472895

Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

PubMed Central

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-01-01

Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies. PMID:19383142

Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

PubMed

Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

2009-04-21

To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease error correction in combination with PIPE cloning. In a sister manuscript we present data on how Gene Composer designed genes and protein constructs can result in improved protein production for structural studies.

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes

PubMed Central

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas. PMID:29872447

Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli

PubMed Central

Evfratov, Sergey A.; Osterman, Ilya A.; Komarova, Ekaterina S.; Pogorelskaya, Alexandra M.; Rubtsova, Maria P.; Zatsepin, Timofei S.; Semashko, Tatiana A.; Kostryukova, Elena S.; Mironov, Andrey A.; Burnaev, Evgeny; Krymova, Ekaterina; Gelfand, Mikhail S.; Govorun, Vadim M.; Bogdanov, Alexey A.; Dontsova, Olga A.

2017-01-01

Abstract Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs. PMID:27899632

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes.

PubMed

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

Prediction of host - pathogen protein interactions between Mycobacterium tuberculosis and Homo sapiens using sequence motifs.

PubMed

Huo, Tong; Liu, Wei; Guo, Yu; Yang, Cheng; Lin, Jianping; Rao, Zihe

2015-03-26

Emergence of multiple drug resistant strains of M. tuberculosis (MDR-TB) threatens to derail global efforts aimed at reigning in the pathogen. Co-infections of M. tuberculosis with HIV are difficult to treat. To counter these new challenges, it is essential to study the interactions between M. tuberculosis and the host to learn how these bacteria cause disease. We report a systematic flow to predict the host pathogen interactions (HPIs) between M. tuberculosis and Homo sapiens based on sequence motifs. First, protein sequences were used as initial input for identifying the HPIs by 'interolog' method. HPIs were further filtered by prediction of domain-domain interactions (DDIs). Functional annotations of protein and publicly available experimental results were applied to filter the remaining HPIs. Using such a strategy, 118 pairs of HPIs were identified, which involve 43 proteins from M. tuberculosis and 48 proteins from Homo sapiens. A biological interaction network between M. tuberculosis and Homo sapiens was then constructed using the predicted inter- and intra-species interactions based on the 118 pairs of HPIs. Finally, a web accessible database named PATH (Protein interactions of M. tuberculosis and Human) was constructed to store these predicted interactions and proteins. This interaction network will facilitate the research on host-pathogen protein-protein interactions, and may throw light on how M. tuberculosis interacts with its host.

Computational Design of DNA-Binding Proteins.

PubMed

Thyme, Summer; Song, Yifan

2016-01-01

Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering.

Efficient encapsulation of proteins with random copolymers.

PubMed

Nguyen, Trung Dac; Qiao, Baofu; Olvera de la Cruz, Monica

2018-06-12

Membraneless organelles are aggregates of disordered proteins that form spontaneously to promote specific cellular functions in vivo. The possibility of synthesizing membraneless organelles out of cells will therefore enable fabrication of protein-based materials with functions inherent to biological matter. Since random copolymers contain various compositions and sequences of solvophobic and solvophilic groups, they are expected to function in nonbiological media similarly to a set of disordered proteins in membraneless organelles. Interestingly, the internal environment of these organelles has been noted to behave more like an organic solvent than like water. Therefore, an adsorbed layer of random copolymers that mimics the function of disordered proteins could, in principle, protect and enhance the proteins' enzymatic activity even in organic solvents, which are ideal when the products and/or the reactants have limited solubility in aqueous media. Here, we demonstrate via multiscale simulations that random copolymers efficiently incorporate proteins into different solvents with the potential to optimize their enzymatic activity. We investigate the key factors that govern the ability of random copolymers to encapsulate proteins, including the adsorption energy, copolymer average composition, and solvent selectivity. The adsorbed polymer chains have remarkably similar sequences, indicating that the proteins are able to select certain sequences that best reduce their exposure to the solvent. We also find that the protein surface coverage decreases when the fluctuation in the average distance between the protein adsorption sites increases. The results herein set the stage for computational design of random copolymers for stabilizing and delivering proteins across multiple media.

Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)

PubMed Central

Pamp, Sünje J.; Harrington, Eoghan D.; Quake, Stephen R.; Relman, David A.; Blainey, Paul C.

2012-01-01

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which “holdfasts” and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract. PMID:22434425

Extreme diversity of scorpion venom peptides and proteins revealed by transcriptomic analysis: implication for proteome evolution of scorpion venom arsenal.

PubMed

Ma, Yibao; He, Yawen; Zhao, Ruiming; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

2012-02-16

Venom is an important genetic development crucial to the survival of scorpions for over 400 million years. We studied the evolution of the scorpion venom arsenal by means of comparative transcriptome analysis of venom glands and phylogenetic analysis of shared types of venom peptides and proteins between buthids and euscorpiids. Fifteen types of venom peptides and proteins were sequenced during the venom gland transcriptome analyses of two Buthidae species (Lychas mucronatus and Isometrus maculatus) and one Euscorpiidae species (Scorpiops margerisonae). Great diversity has been observed in translated amino acid sequences of these transcripts for venom peptides and proteins. Seven types of venom peptides and proteins were shared between buthids and euscorpiids. Molecular phylogenetic analysis revealed that at least five of the seven common types of venom peptides and proteins were likely recruited into the scorpion venom proteome before the lineage split between Buthidae and Euscorpiidae with their corresponding genes undergoing individual or multiple gene duplication events. These are α-KTxs, βKSPNs (β-KTxs and scorpines), anionic peptides, La1-like peptides, and SPSVs (serine proteases from scorpion venom). Multiple types of venom peptides and proteins were demonstrated to be continuously recruited into the venom proteome during the evolution process of individual scorpion lineages. Our results provide an insight into the recruitment pattern of the scorpion venom arsenal for the first time. Copyright © 2011 Elsevier B.V. All rights reserved.

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Characterization and production of multifunctional cationic peptides derived from rice proteins.

PubMed

Taniguchi, Masayuki; Ochiai, Akihito

2017-04-01

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.

Resolving the multiple sequence alignment problem using biogeography-based optimization with multiple populations.

PubMed

Zemali, El-Amine; Boukra, Abdelmadjid

2015-08-01

The multiple sequence alignment (MSA) is one of the most challenging problems in bioinformatics, it involves discovering similarity between a set of protein or DNA sequences. This paper introduces a new method for the MSA problem called biogeography-based optimization with multiple populations (BBOMP). It is based on a recent metaheuristic inspired from the mathematics of biogeography named biogeography-based optimization (BBO). To improve the exploration ability of BBO, we have introduced a new concept allowing better exploration of the search space. It consists of manipulating multiple populations having each one its own parameters. These parameters are used to build up progressive alignments allowing more diversity. At each iteration, the best found solution is injected in each population. Moreover, to improve solution quality, six operators are defined. These operators are selected with a dynamic probability which changes according to the operators efficiency. In order to test proposed approach performance, we have considered a set of datasets from Balibase 2.0 and compared it with many recent algorithms such as GAPAM, MSA-GA, QEAMSA and RBT-GA. The results show that the proposed approach achieves better average score than the previously cited methods.

ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species.

PubMed

Zeng, Victor; Extavour, Cassandra G

2012-01-01

The increased throughput and decreased cost of next-generation sequencing (NGS) have shifted the bottleneck genomic research from sequencing to annotation, analysis and accessibility. This is particularly challenging for research communities working on organisms that lack the basic infrastructure of a sequenced genome, or an efficient way to utilize whatever sequence data may be available. Here we present a new database, the Assembled Searchable Giant Arthropod Read Database (ASGARD). This database is a repository and search engine for transcriptomic data from arthropods that are of high interest to multiple research communities but currently lack sequenced genomes. We demonstrate the functionality and utility of ASGARD using de novo assembled transcriptomes from the milkweed bug Oncopeltus fasciatus, the cricket Gryllus bimaculatus and the amphipod crustacean Parhyale hawaiensis. We have annotated these transcriptomes to assign putative orthology, coding region determination, protein domain identification and Gene Ontology (GO) term annotation to all possible assembly products. ASGARD allows users to search all assemblies by orthology annotation, GO term annotation or Basic Local Alignment Search Tool. User-friendly features of ASGARD include search term auto-completion suggestions based on database content, the ability to download assembly product sequences in FASTA format, direct links to NCBI data for predicted orthologs and graphical representation of the location of protein domains and matches to similar sequences from the NCBI non-redundant database. ASGARD will be a useful repository for transcriptome data from future NGS studies on these and other emerging model arthropods, regardless of sequencing platform, assembly or annotation status. This database thus provides easy, one-stop access to multi-species annotated transcriptome information. We anticipate that this database will be useful for members of multiple research communities, including developmental biology, physiology, evolutionary biology, ecology, comparative genomics and phylogenomics. Database URL: asgard.rc.fas.harvard.edu.

Multistate approaches in computational protein design

PubMed Central

Davey, James A; Chica, Roberto A

2012-01-01

Computational protein design (CPD) is a useful tool for protein engineers. It has been successfully applied towards the creation of proteins with increased thermostability, improved binding affinity, novel enzymatic activity, and altered ligand specificity. Traditionally, CPD calculations search and rank sequences using a single fixed protein backbone template in an approach referred to as single-state design (SSD). While SSD has enjoyed considerable success, certain design objectives require the explicit consideration of multiple conformational and/or chemical states. Cases where a “multistate” approach may be advantageous over the SSD approach include designing conformational changes into proteins, using native ensembles to mimic backbone flexibility, and designing ligand or oligomeric association specificities. These design objectives can be efficiently tackled using multistate design (MSD), an emerging methodology in CPD that considers any number of protein conformational or chemical states as inputs instead of a single protein backbone template, as in SSD. In this review article, recent examples of the successful design of a desired property into proteins using MSD are described. These studies employing MSD are divided into two categories—those that utilized multiple conformational states, and those that utilized multiple chemical states. In addition, the scoring of competing states during negative design is discussed as a current challenge for MSD. PMID:22811394

Multi-Harmony: detecting functional specificity from sequence alignment

PubMed Central

Brandt, Bernd W.; Feenstra, K. Anton; Heringa, Jaap

2010-01-01

Many protein families contain sub-families with functional specialization, such as binding different ligands or being involved in different protein–protein interactions. A small number of amino acids generally determine functional specificity. The identification of these residues can aid the understanding of protein function and help finding targets for experimental analysis. Here, we present multi-Harmony, an interactive web sever for detecting sub-type-specific sites in proteins starting from a multiple sequence alignment. Combining our Sequence Harmony (SH) and multi-Relief (mR) methods in one web server allows simultaneous analysis and comparison of specificity residues; furthermore, both methods have been significantly improved and extended. SH has been extended to cope with more than two sub-groups. mR has been changed from a sampling implementation to a deterministic one, making it more consistent and user friendly. For both methods Z-scores are reported. The multi-Harmony web server produces a dynamic output page, which includes interactive connections to the Jalview and Jmol applets, thereby allowing interactive analysis of the results. Multi-Harmony is available at http://www.ibi.vu.nl/ programs/shmrwww. PMID:20525785

Ferritins: dynamic management of biological iron and oxygen chemistry.

PubMed

Liu, Xiaofeng; Theil, Elizabeth C

2005-03-01

Ferritins are spherical, cage-like proteins with nanocavities formed by multiple polypeptide subunits (four-helix bundles) that manage iron/oxygen chemistry. Catalytic coupling yields diferric oxo/hydroxo complexes at ferroxidase sites in maxi-ferritin subunits (24 subunits, 480 kDa; plants, animals, microorganisms). Oxidation occurs at the cavity surface of mini-ferritins/Dps proteins (12 subunits, 240 kDa; bacteria). Oxidation products are concentrated as minerals in the nanocavity for iron-protein cofactor synthesis (maxi-ferritins) or DNA protection (mini-ferritins). The protein cage and nanocavity characterize all ferritins, although amino acid sequences diverge, especially in bacteria. Catalytic oxidation/di-iron coupling in the protein cage (maxi-ferritins, 480 kDa; plants, bacteria and animal cell-specific isoforms) or on the cavity surface (mini-ferritins/Dps proteins, 280 kDa; bacteria) initiates mineralization. Gated pores (eight or four), symmetrically arranged, control iron flow. The multiple ferritin functions combine pore, channel, and catalytic functions in compact protein structures required for life and disease response.

Sequence analysis of serum albumins reveals the molecular evolution of ligand recognition properties.

PubMed

Fanali, Gabriella; Ascenzi, Paolo; Bernardi, Giorgio; Fasano, Mauro

2012-01-01

Serum albumin (SA) is a circulating protein providing a depot and carrier for many endogenous and exogenous compounds. At least seven major binding sites have been identified by structural and functional investigations mainly in human SA. SA is conserved in vertebrates, with at least 49 entries in protein sequence databases. The multiple sequence analysis of this set of entries leads to the definition of a cladistic tree for the molecular evolution of SA orthologs in vertebrates, thus showing the clustering of the considered species, with lamprey SAs (Lethenteron japonicum and Petromyzon marinus) in a separate outgroup. Sequence analysis aimed at searching conserved domains revealed that most SA sequences are made up by three repeated domains (about 600 residues), as extensively characterized for human SA. On the contrary, lamprey SAs are giant proteins (about 1400 residues) comprising seven repeated domains. The phylogenetic analysis of the SA family reveals a stringent correlation with the taxonomic classification of the species available in sequence databases. A focused inspection of the sequences of ligand binding sites in SA revealed that in all sites most residues involved in ligand binding are conserved, although the versatility towards different ligands could be peculiar of higher organisms. Moreover, the analysis of molecular links between the different sites suggests that allosteric modulation mechanisms could be restricted to higher vertebrates.

KinView: A visual comparative sequence analysis tool for integrated kinome research

PubMed Central

McSkimming, Daniel Ian; Dastgheib, Shima; Baffi, Timothy R.; Byrne, Dominic P.; Ferries, Samantha; Scott, Steven Thomas; Newton, Alexandra C.; Eyers, Claire E.; Kochut, Krzysztof J.; Eyers, Patrick A.

2017-01-01

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats. PMID:27731453

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arakaki, Tracy; Le Trong, Isolde; Structural Genomics of Pathogenic Protozoa

2006-03-01

The crystal structure of a conserved hypothetical protein from L. major, Pfam sequence family PF04543, structural genomics target ID Lmaj006129AAA, has been determined at a resolution of 1.6 Å. The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD)more » using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (R{sub free} = 0.229) at 1.60 Å resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.« less

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Population entropies estimates of proteins

NASA Astrophysics Data System (ADS)

Low, Wai Yee

2017-05-01

The Shannon entropy equation provides a way to estimate variability of amino acids sequences in a multiple sequence alignment of proteins. Knowledge of protein variability is useful in many areas such as vaccine design, identification of antibody binding sites, and exploration of protein 3D structural properties. In cases where the population entropies of a protein are of interest but only a small sample size can be obtained, a method based on linear regression and random subsampling can be used to estimate the population entropy. This method is useful for comparisons of entropies where the actual sequence counts differ and thus, correction for alignment size bias is needed. In the current work, an R based package named EntropyCorrect that enables estimation of population entropy is presented and an empirical study on how well this new algorithm performs on simulated dataset of various combinations of population and sample sizes is discussed. The package is available at https://github.com/lloydlow/EntropyCorrect. This article, which was originally published online on 12 May 2017, contained an error in Eq. (1), where the summation sign was missing. The corrected equation appears in the Corrigendum attached to the pdf.

ZifBASE: a database of zinc finger proteins and associated resources.

PubMed

Jayakanthan, Mannu; Muthukumaran, Jayaraman; Chandrasekar, Sanniyasi; Chawla, Konika; Punetha, Ankita; Sundar, Durai

2009-09-09

Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative. A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/zifbase.

DUF1220 protein domains drive proliferation in human neural stem cells and are associated with increased cortical volume in anthropoid primates.

PubMed

Keeney, J G; Davis, J M; Siegenthaler, J; Post, M D; Nielsen, B S; Hopkins, W D; Sikela, J M

2015-09-01

Genome sequences encoding DUF1220 protein domains show a burst in copy number among anthropoid species and especially humans, where they have undergone the greatest human lineage-specific copy number expansion of any protein coding sequence in the genome. While DUF1220 copy number shows a dosage-related association with brain size in both normal populations and in 1q21.1-associated microcephaly and macrocephaly, a function for these domains has not yet been described. Here we provide multiple lines of evidence supporting the view that DUF1220 domains function as drivers of neural stem cell proliferation among anthropoid species including humans. First, we show that brain MRI data from 131 individuals across 7 anthropoid species shows a strong correlation between DUF1220 copy number and multiple brain size-related measures. Using in situ hybridization analyses of human fetal brain, we also show that DUF1220 domains are expressed in the ventricular zone and primarily during human cortical neurogenesis, and are therefore expressed at the right time and place to be affecting cortical brain development. Finally, we demonstrate that in vitro expression of DUF1220 sequences in neural stem cells strongly promotes proliferation. Taken together, these data provide the strongest evidence so far reported implicating DUF1220 dosage in anthropoid and human brain expansion through mechanisms involving increasing neural stem cell proliferation.

Verification of 2A peptide cleavage.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

PubMed

Fu, Lijuan; Shi, Zhimin; Luo, Guanzheng; Tu, Weihong; Wang, XiuJie; Fang, Zhide; Li, XiaoChing

2014-10-01

Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

BIPAD: A web server for modeling bipartite sequence elements

PubMed Central

Bi, Chengpeng; Rogan, Peter K

2006-01-01

Background Many dimeric protein complexes bind cooperatively to families of bipartite nucleic acid sequence elements, which consist of pairs of conserved half-site sequences separated by intervening distances that vary among individual sites. Results We introduce the Bipad Server [1], a web interface to predict sequence elements embedded within unaligned sequences. Either a bipartite model, consisting of a pair of one-block position weight matrices (PWM's) with a gap distribution, or a single PWM matrix for contiguous single block motifs may be produced. The Bipad program performs multiple local alignment by entropy minimization and cyclic refinement using a stochastic greedy search strategy. The best models are refined by maximizing incremental information contents among a set of potential models with varying half site and gap lengths. Conclusion The web service generates information positional weight matrices, identifies binding site motifs, graphically represents the set of discovered elements as a sequence logo, and depicts the gap distribution as a histogram. Server performance was evaluated by generating a collection of bipartite models for distinct DNA binding proteins. PMID:16503993

Integration of promoters, inverted repeat sequences and proteomic data into a model for high silencing efficiency of coeliac disease related gliadins in bread wheat

PubMed Central

2013-01-01

Background Wheat gluten has unique nutritional and technological characteristics, but is also a major trigger of allergies and intolerances. One of the most severe diseases caused by gluten is coeliac disease. The peptides produced in the digestive tract by the incomplete digestion of gluten proteins trigger the disease. The majority of the epitopes responsible reside in the gliadin fraction of gluten. The location of the multiple gliadin genes in blocks has to date complicated their elimination by classical breeding techniques or by the use of biotechnological tools. As an approach to silence multiple gliadin genes we have produced 38 transgenic lines of bread wheat containing combinations of two endosperm-specific promoters and three different inverted repeat sequences to silence three fractions of gliadins by RNA interference. Results The effects of the RNA interference constructs on the content of the gluten proteins, total protein and starch, thousand seed weights and SDSS quality tests of flour were analyzed in these transgenic lines in two consecutive years. The characteristics of the inverted repeat sequences were the main factor that determined the efficiency of silencing. The promoter used had less influence on silencing, although a synergy in silencing efficiency was observed when the two promoters were used simultaneously. Genotype and the environment also influenced silencing efficiency. Conclusions We conclude that to obtain wheat lines with an optimum reduction of toxic gluten epitopes one needs to take into account the factors of inverted repeat sequences design, promoter choice and also the wheat background used. PMID:24044767

Alga-PrAS (Algal Protein Annotation Suite): A Database of Comprehensive Annotation in Algal Proteomes

PubMed Central

Kurotani, Atsushi; Yamada, Yutaka

2017-01-01

Algae are smaller organisms than land plants and offer clear advantages in research over terrestrial species in terms of rapid production, short generation time and varied commercial applications. Thus, studies investigating the practical development of effective algal production are important and will improve our understanding of both aquatic and terrestrial plants. In this study we estimated multiple physicochemical and secondary structural properties of protein sequences, the predicted presence of post-translational modification (PTM) sites, and subcellular localization using a total of 510,123 protein sequences from the proteomes of 31 algal and three plant species. Algal species were broadly selected from green and red algae, glaucophytes, oomycetes, diatoms and other microalgal groups. The results were deposited in the Algal Protein Annotation Suite database (Alga-PrAS; http://alga-pras.riken.jp/), which can be freely accessed online. PMID:28069893

DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules.

PubMed

Eernisse, D J

1992-04-01

DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers. They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing. DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list. Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs. Single or multiple sequences can be manipulated to aid in phylogenetic analysis. Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols. Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis. Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package. Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences. DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations. Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest. The complete package is available via anonymous ftp and is free for non-commercial uses.

Query-seeded iterative sequence similarity searching improves selectivity 5–20-fold

PubMed Central

Li, Weizhong; Lopez, Rodrigo

2017-01-01

Abstract Iterative similarity search programs, like psiblast, jackhmmer, and psisearch, are much more sensitive than pairwise similarity search methods like blast and ssearch because they build a position specific scoring model (a PSSM or HMM) that captures the pattern of sequence conservation characteristic to a protein family. But models are subject to contamination; once an unrelated sequence has been added to the model, homologs of the unrelated sequence will also produce high scores, and the model can diverge from the original protein family. Examination of alignment errors during psiblast PSSM contamination suggested a simple strategy for dramatically reducing PSSM contamination. psiblast PSSMs are built from the query-based multiple sequence alignment (MSA) implied by the pairwise alignments between the query model (PSSM, HMM) and the subject sequences in the library. When the original query sequence residues are inserted into gapped positions in the aligned subject sequence, the resulting PSSM rarely produces alignment over-extensions or alignments to unrelated sequences. This simple step, which tends to anchor the PSSM to the original query sequence and slightly increase target percent identity, can reduce the frequency of false-positive alignments more than 20-fold compared with psiblast and jackhmmer, with little loss in search sensitivity. PMID:27923999

Integrating multiple genomic data to predict disease-causing nonsynonymous single nucleotide variants in exome sequencing studies.

PubMed

Wu, Jiaxin; Li, Yanda; Jiang, Rui

2014-03-01

Exome sequencing has been widely used in detecting pathogenic nonsynonymous single nucleotide variants (SNVs) for human inherited diseases. However, traditional statistical genetics methods are ineffective in analyzing exome sequencing data, due to such facts as the large number of sequenced variants, the presence of non-negligible fraction of pathogenic rare variants or de novo mutations, and the limited size of affected and normal populations. Indeed, prevalent applications of exome sequencing have been appealing for an effective computational method for identifying causative nonsynonymous SNVs from a large number of sequenced variants. Here, we propose a bioinformatics approach called SPRING (Snv PRioritization via the INtegration of Genomic data) for identifying pathogenic nonsynonymous SNVs for a given query disease. Based on six functional effect scores calculated by existing methods (SIFT, PolyPhen2, LRT, MutationTaster, GERP and PhyloP) and five association scores derived from a variety of genomic data sources (gene ontology, protein-protein interactions, protein sequences, protein domain annotations and gene pathway annotations), SPRING calculates the statistical significance that an SNV is causative for a query disease and hence provides a means of prioritizing candidate SNVs. With a series of comprehensive validation experiments, we demonstrate that SPRING is valid for diseases whose genetic bases are either partly known or completely unknown and effective for diseases with a variety of inheritance styles. In applications of our method to real exome sequencing data sets, we show the capability of SPRING in detecting causative de novo mutations for autism, epileptic encephalopathies and intellectual disability. We further provide an online service, the standalone software and genome-wide predictions of causative SNVs for 5,080 diseases at http://bioinfo.au.tsinghua.edu.cn/spring.

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

PubMed Central

del Val, Coral; White, Stephen H.

2014-01-01

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

Analysis of ER Resident Proteins in S. cerevisiae: Implementation of H/KDEL Retrieval Sequences

PubMed Central

Young, Carissa L.; Raden, David L.; Robinson, Anne S.

2013-01-01

An elaborate quality control system regulates ER homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high-throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of PCR-based modules including codon-optimized epitopes and FP variants complete with C-terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins. To monitor multiple proteins simultaneously, H/KDEL cassettes are available with six different selection markers, providing optimal flexibility for live-cell imaging and multicolor labeling in vivo. A single pair of PCR primers can be used for the amplification of these 26 modules, enabling numerous combinations of tags and selection markers. The versatility of pCY H/KDEL cassettes was demonstrated by labeling BiP/Kar2p, Pdi1p, and Scj1p with all novel tags, thus providing a direct comparison among FP variants. Furthermore, to advance in vitro studies of yeast ER proteins, Strep-tag II was engineered with a C-terminal retrieval sequence. Here, an efficient purification strategy was established for BiP under physiological conditions. PMID:23324027

Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans

PubMed Central

Romeo, Stefano; Yin, Wu; Kozlitina, Julia; Pennacchio, Len A.; Boerwinkle, Eric; Hobbs, Helen H.; Cohen, Jonathan C.

2008-01-01

The relative activity of lipoprotein lipase (LPL) in different tissues controls the partitioning of lipoprotein-derived fatty acids between sites of fat storage (adipose tissue) and oxidation (heart and skeletal muscle). Here we used a reverse genetic strategy to test the hypothesis that 4 angiopoietin-like proteins (ANGPTL3, -4, -5, and -6) play key roles in triglyceride (TG) metabolism in humans. We re-sequenced the coding regions of the genes encoding these proteins and identified multiple rare nonsynonymous (NS) sequence variations that were associated with low plasma TG levels but not with other metabolic phenotypes. Functional studies revealed that all mutant alleles of ANGPTL3 and ANGPTL4 that were associated with low plasma TG levels interfered either with the synthesis or secretion of the protein or with the ability of the ANGPTL protein to inhibit LPL. A total of 1% of the Dallas Heart Study population and 4% of those participants with a plasma TG in the lowest quartile had a rare loss-of-function mutation in ANGPTL3, ANGPTL4, or ANGPTL5. Thus, ANGPTL3, ANGPTL4, and ANGPTL5, but not ANGPTL6, play nonredundant roles in TG metabolism, and multiple alleles at these loci cumulatively contribute to variability in plasma TG levels in humans. PMID:19075393

Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

PubMed

Zhu, Ruo-Lin; Lei, Xiao-Ying; Ke, Fei; Yuan, Xiu-Ping; Zhang, Qi-Ya

2011-02-01

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size. Copyright © 2010 Elsevier B.V. All rights reserved.

Applying the Concept of Peptide Uniqueness to Anti-Polio Vaccination

PubMed Central

Kanduc, Darja; Fasano, Candida; Capone, Giovanni; Pesce Delfino, Antonella; Calabrò, Michele; Polimeno, Lorenzo

2015-01-01

Background. Although rare, adverse events may associate with anti-poliovirus vaccination thus possibly hampering global polio eradication worldwide. Objective. To design peptide-based anti-polio vaccines exempt from potential cross-reactivity risks and possibly able to reduce rare potential adverse events such as the postvaccine paralytic poliomyelitis due to the tendency of the poliovirus genome to mutate. Methods. Proteins from poliovirus type 1, strain Mahoney, were analyzed for amino acid sequence identity to the human proteome at the pentapeptide level, searching for sequences that (1) have zero percent of identity to human proteins, (2) are potentially endowed with an immunologic potential, and (3) are highly conserved among poliovirus strains. Results. Sequence analyses produced a set of consensus epitopic peptides potentially able to generate specific anti-polio immune responses exempt from cross-reactivity with the human host. Conclusion. Peptide sequences unique to poliovirus proteins and conserved among polio strains might help formulate a specific and universal anti-polio vaccine able to react with multiple viral strains and exempt from the burden of possible cross-reactions with human proteins. As an additional advantage, using a peptide-based vaccine instead of current anti-polio DNA vaccines would eliminate the rare post-polio poliomyelitis cases and other disabling symptoms that may appear following vaccination. PMID:26568962

Bioinformatics prediction of siRNAs as potential antiviral agents against dengue viruses

PubMed Central

Villegas-Rosales, Paula M; Méndez-Tenorio, Alfonso; Ortega-Soto, Elizabeth; Barrón, Blanca L

2012-01-01

Dengue virus (DENV 1-4) represents the major emerging arthropod-borne viral infection in the world. Currently, there is neither an available vaccine nor a specific treatment. Hence, there is a need of antiviral drugs for these viral infections; we describe the prediction of short interfering RNA (siRNA) as potential therapeutic agents against the four DENV serotypes. Our strategy was to carry out a series of multiple alignments using ClustalX program to find conserved sequences among the four DENV serotype genomes to obtain a consensus sequence for siRNAs design. A highly conserved sequence among the four DENV serotypes, located in the encoding sequence for NS4B and NS5 proteins was found. A total of 2,893 complete DENV genomes were downloaded from the NCBI, and after a depuration procedure to identify identical sequences, 220 complete DENV genomes were left. They were edited to select the NS4B and NS5 sequences, which were aligned to obtain a consensus sequence. Three different servers were used for siRNA design, and the resulting siRNAs were aligned to identify the most prevalent sequences. Three siRNAs were chosen, one targeted the genome region that codifies for NS4B protein and the other two; the region for NS5 protein. Predicted secondary structure for DENV genomes was used to demonstrate that the siRNAs were able to target the viral genome forming double stranded structures, necessary to activate the RNA silencing machinery. PMID:22829722

SubVis: an interactive R package for exploring the effects of multiple substitution matrices on pairwise sequence alignment

PubMed Central

Coan, Heather B.; Youker, Robert T.

2017-01-01

Understanding how proteins mutate is critical to solving a host of biological problems. Mutations occur when an amino acid is substituted for another in a protein sequence. The set of likelihoods for amino acid substitutions is stored in a matrix and input to alignment algorithms. The quality of the resulting alignment is used to assess the similarity of two or more sequences and can vary according to assumptions modeled by the substitution matrix. Substitution strategies with minor parameter variations are often grouped together in families. For example, the BLOSUM and PAM matrix families are commonly used because they provide a standard, predefined way of modeling substitutions. However, researchers often do not know if a given matrix family or any individual matrix within a family is the most suitable. Furthermore, predefined matrix families may inaccurately reflect a particular hypothesis that a researcher wishes to model or otherwise result in unsatisfactory alignments. In these cases, the ability to compare the effects of one or more custom matrices may be needed. This laborious process is often performed manually because the ability to simultaneously load multiple matrices and then compare their effects on alignments is not readily available in current software tools. This paper presents SubVis, an interactive R package for loading and applying multiple substitution matrices to pairwise alignments. Users can simultaneously explore alignments resulting from multiple predefined and custom substitution matrices. SubVis utilizes several of the alignment functions found in R, a common language among protein scientists. Functions are tied together with the Shiny platform which allows the modification of input parameters. Information regarding alignment quality and individual amino acid substitutions is displayed with the JavaScript language which provides interactive visualizations for revealing both high-level and low-level alignment information. PMID:28674656

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

PubMed

Tong, Zheng; Wang, Dan; Sun, Yong; Yang, Qian; Meng, Xueru; Wang, Limin; Feng, Weiqiang; Li, Ling; Wurtele, Eve Syrkin; Wang, Xuchu

2017-05-02

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF 138,175,258 and SRPP 117,204,243 , were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF 258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF 175 or REF 258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

ADOMA: A Command Line Tool to Modify ClustalW Multiple Alignment Output.

PubMed

Zaal, Dionne; Nota, Benjamin

2016-01-01

We present ADOMA, a command line tool that produces alternative outputs from ClustalW multiple alignments of nucleotide or protein sequences. ADOMA can simplify the output of alignments by showing only the different residues between sequences, which is often desirable when only small differences such as single nucleotide polymorphisms are present (e.g., between different alleles). Another feature of ADOMA is that it can enhance the ClustalW output by coloring the residues in the alignment. This tool is easily integrated into automated Linux pipelines for next-generation sequencing data analysis, and may be useful for researchers in a broad range of scientific disciplines including evolutionary biology and biomedical sciences. The source code is freely available at https://sourceforge. net/projects/adoma/. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Network Biomarkers of Bladder Cancer Based on a Genome-Wide Genetic and Epigenetic Network Derived from Next-Generation Sequencing Data.

PubMed

Li, Cheng-Wei; Chen, Bor-Sen

2016-01-01

Epigenetic and microRNA (miRNA) regulation are associated with carcinogenesis and the development of cancer. By using the available omics data, including those from next-generation sequencing (NGS), genome-wide methylation profiling, candidate integrated genetic and epigenetic network (IGEN) analysis, and drug response genome-wide microarray analysis, we constructed an IGEN system based on three coupling regression models that characterize protein-protein interaction networks (PPINs), gene regulatory networks (GRNs), miRNA regulatory networks (MRNs), and epigenetic regulatory networks (ERNs). By applying system identification method and principal genome-wide network projection (PGNP) to IGEN analysis, we identified the core network biomarkers to investigate bladder carcinogenic mechanisms and design multiple drug combinations for treating bladder cancer with minimal side-effects. The progression of DNA repair and cell proliferation in stage 1 bladder cancer ultimately results not only in the derepression of miR-200a and miR-200b but also in the regulation of the TNF pathway to metastasis-related genes or proteins, cell proliferation, and DNA repair in stage 4 bladder cancer. We designed a multiple drug combination comprising gefitinib, estradiol, yohimbine, and fulvestrant for treating stage 1 bladder cancer with minimal side-effects, and another multiple drug combination comprising gefitinib, estradiol, chlorpromazine, and LY294002 for treating stage 4 bladder cancer with minimal side-effects.

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

PubMed

Degenhardt, Jörg; Köllner, Tobias G; Gershenzon, Jonathan

2009-01-01

The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th‐century pandemics

PubMed Central

Pasricha, Gunisha; Mishra, Akhilesh C.; Chakrabarti, Alok K.

2012-01-01

Please cite this paper as: Pasricha et al. (2012) Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the Influenza A virus subtypes responsible for the 20th‐century pandemics. Influenza and Other Respiratory Viruses 7(4), 497–505. Background  PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Methods  Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Results  Analysis showed that 96·4% of the H5N1 influenza viruses harbored full‐length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th‐century pandemic influenza viruses contained full‐length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human‐ and avian host‐specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Conclusions  Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host‐specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity. PMID:22788742

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

PreSSAPro: a software for the prediction of secondary structure by amino acid properties.

PubMed

Costantini, Susan; Colonna, Giovanni; Facchiano, Angelo M

2007-10-01

PreSSAPro is a software, available to the scientific community as a free web service designed to provide predictions of secondary structures starting from the amino acid sequence of a given protein. Predictions are based on our recently published work on the amino acid propensities for secondary structures in either large but not homogeneous protein data sets, as well as in smaller but homogeneous data sets corresponding to protein structural classes, i.e. all-alpha, all-beta, or alpha-beta proteins. Predictions result improved by the use of propensities evaluated for the right protein class. PreSSAPro predicts the secondary structure according to the right protein class, if known, or gives a multiple prediction with reference to the different structural classes. The comparison of these predictions represents a novel tool to evaluate what sequence regions can assume different secondary structures depending on the structural class assignment, in the perspective of identifying proteins able to fold in different conformations. The service is available at the URL http://bioinformatica.isa.cnr.it/PRESSAPRO/.

The Multiple-Minima Problem in Protein Folding

NASA Astrophysics Data System (ADS)

Scheraga, Harold A.

1991-10-01

The conformational energy surface of a polypeptide or protein has many local minima, and conventional energy minimization procedures reach only a local minimum (near the starting point of the optimization algorithm) instead of the global minimum (the multiple-minima problem). Several procedures have been developed to surmount this problem, the most promising of which are: (a) build up procedure, (b) optimization of electrostatics, (c) Monte Carlo-plus-energy minimization, (d) electrostatically-driven Monte Carlo, (e) inclusion of distance restraints, (f) adaptive importance-sampling Monte Carlo, (g) relaxation of dimensionality, (h) pattern-recognition, and (i) diffusion equation method. These procedures have been applied to a variety of polypeptide structural problems, and the results of such computations are presented. These include the computation of the structures of open-chain and cyclic peptides, fibrous proteins and globular proteins. Present efforts are being devoted to scaling up these procedures from small polypeptides to proteins, to try to compute the three-dimensional structure of a protein from its amino sequence.

A force-based, parallel assay for the quantification of protein-DNA interactions.

PubMed

Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

2014-01-01

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications.

PubMed

Buerth, Christoph; Tielker, Denis; Ernst, Joachim F

2016-08-01

The yeast Candida utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C. utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C. utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C. utilis and its presumed parent Cyberlindnera (Pichia) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C. jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C. utilis, as well as current and future biotechnological and medical applications of C. utilis and C. jadinii.

The ConSurf-DB: pre-calculated evolutionary conservation profiles of protein structures.

PubMed

Goldenberg, Ofir; Erez, Elana; Nimrod, Guy; Ben-Tal, Nir

2009-01-01

ConSurf-DB is a repository for evolutionary conservation analysis of the proteins of known structures in the Protein Data Bank (PDB). Sequence homologues of each of the PDB entries were collected and aligned using standard methods. The evolutionary conservation of each amino acid position in the alignment was calculated using the Rate4Site algorithm, implemented in the ConSurf web server. The algorithm takes into account the phylogenetic relations between the aligned proteins and the stochastic nature of the evolutionary process explicitly. Rate4Site assigns a conservation level for each position in the multiple sequence alignment using an empirical Bayesian inference. Visual inspection of the conservation patterns on the 3D structure often enables the identification of key residues that comprise the functionally important regions of the protein. The repository is updated with the latest PDB entries on a monthly basis and will be rebuilt annually. ConSurf-DB is available online at http://consurfdb.tau.ac.il/

The ConSurf-DB: pre-calculated evolutionary conservation profiles of protein structures

PubMed Central

Goldenberg, Ofir; Erez, Elana; Nimrod, Guy; Ben-Tal, Nir

2009-01-01

ConSurf-DB is a repository for evolutionary conservation analysis of the proteins of known structures in the Protein Data Bank (PDB). Sequence homologues of each of the PDB entries were collected and aligned using standard methods. The evolutionary conservation of each amino acid position in the alignment was calculated using the Rate4Site algorithm, implemented in the ConSurf web server. The algorithm takes into account the phylogenetic relations between the aligned proteins and the stochastic nature of the evolutionary process explicitly. Rate4Site assigns a conservation level for each position in the multiple sequence alignment using an empirical Bayesian inference. Visual inspection of the conservation patterns on the 3D structure often enables the identification of key residues that comprise the functionally important regions of the protein. The repository is updated with the latest PDB entries on a monthly basis and will be rebuilt annually. ConSurf-DB is available online at http://consurfdb.tau.ac.il/ PMID:18971256

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-09-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease

PubMed Central

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-01-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes. PMID:25333063

Improving homology modeling of G-protein coupled receptors through multiple-template derived conserved inter-residue interactions

NASA Astrophysics Data System (ADS)

Chaudhari, Rajan; Heim, Andrew J.; Li, Zhijun

2015-05-01

Evidenced by the three-rounds of G-protein coupled receptors (GPCR) Dock competitions, improving homology modeling methods of helical transmembrane proteins including the GPCRs, based on templates of low sequence identity, remains an eminent challenge. Current approaches addressing this challenge adopt the philosophy of "modeling first, refinement next". In the present work, we developed an alternative modeling approach through the novel application of available multiple templates. First, conserved inter-residue interactions are derived from each additional template through conservation analysis of each template-target pairwise alignment. Then, these interactions are converted into distance restraints and incorporated in the homology modeling process. This approach was applied to modeling of the human β2 adrenergic receptor using the bovin rhodopsin and the human protease-activated receptor 1 as templates and improved model quality was demonstrated compared to the homology model generated by standard single-template and multiple-template methods. This method of "refined restraints first, modeling next", provides a fast and complementary way to the current modeling approaches. It allows rational identification and implementation of additional conserved distance restraints extracted from multiple templates and/or experimental data, and has the potential to be applicable to modeling of all helical transmembrane proteins.

Staufen1 senses overall transcript secondary structure to regulate translation

PubMed Central

Ricci, Emiliano P; Kucukural, Alper; Cenik, Can; Mercier, Blandine C; Singh, Guramrit; Heyer, Erin E; Ashar-Patel, Ami; Peng, Lingtao; Moore, Melissa J

2015-01-01

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3′ untranslated regions (UTRs) or in ‘strongly distal’ 3′ UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3′ UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins. PMID:24336223

Inferring HIV Escape Rates from Multi-Locus Genotype Data

DOE PAGES

Kessinger, Taylor A.; Perelson, Alan S.; Neher, Richard A.

2013-09-03

Cytotoxic T-lymphocytes (CTLs) recognize viral protein fragments displayed by major histocompatibility complex molecules on the surface of virally infected cells and generate an anti-viral response that can kill the infected cells. Virus variants whose protein fragments are not efficiently presented on infected cells or whose fragments are presented but not recognized by CTLs therefore have a competitive advantage and spread rapidly through the population. We present a method that allows a more robust estimation of these escape rates from serially sampled sequence data. The proposed method accounts for competition between multiple escapes by explicitly modeling the accumulation of escape mutationsmore » and the stochastic effects of rare multiple mutants. Applying our method to serially sampled HIV sequence data, we estimate rates of HIV escape that are substantially larger than those previously reported. The method can be extended to complex escapes that require compensatory mutations. We expect our method to be applicable in other contexts such as cancer evolution where time series data is also available.« less

LymPHOS 2.0: an update of a phosphosite database of primary human T cells

PubMed Central

Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

2015-01-01

LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org. PMID:26708986

Genepleio software for effective estimation of gene pleiotropy from protein sequences.

PubMed

Chen, Wenhai; Chen, Dandan; Zhao, Ming; Zou, Yangyun; Zeng, Yanwu; Gu, Xun

2015-01-01

Though pleiotropy, which refers to the phenomenon of a gene affecting multiple traits, has long played a central role in genetics, development, and evolution, estimation of the number of pleiotropy components remains a hard mission to accomplish. In this paper, we report a newly developed software package, Genepleio, to estimate the effective gene pleiotropy from phylogenetic analysis of protein sequences. Since this estimate can be interpreted as the minimum pleiotropy of a gene, it is used to play a role of reference for many empirical pleiotropy measures. This work would facilitate our understanding of how gene pleiotropy affects the pattern of genotype-phenotype map and the consequence of organismal evolution.

GenoMycDB: a database for comparative analysis of mycobacterial genes and genomes.

PubMed

Catanho, Marcos; Mascarenhas, Daniel; Degrave, Wim; Miranda, Antonio Basílio de

2006-03-31

Several databases and computational tools have been created with the aim of organizing, integrating and analyzing the wealth of information generated by large-scale sequencing projects of mycobacterial genomes and those of other organisms. However, with very few exceptions, these databases and tools do not allow for massive and/or dynamic comparison of these data. GenoMycDB (http://www.dbbm.fiocruz.br/GenoMycDB) is a relational database built for large-scale comparative analyses of completely sequenced mycobacterial genomes, based on their predicted protein content. Its central structure is composed of the results obtained after pair-wise sequence alignments among all the predicted proteins coded by the genomes of six mycobacteria: Mycobacterium tuberculosis (strains H37Rv and CDC1551), M. bovis AF2122/97, M. avium subsp. paratuberculosis K10, M. leprae TN, and M. smegmatis MC2 155. The database stores the computed similarity parameters of every aligned pair, providing for each protein sequence the predicted subcellular localization, the assigned cluster of orthologous groups, the features of the corresponding gene, and links to several important databases. Tables containing pairs or groups of potential homologs between selected species/strains can be produced dynamically by user-defined criteria, based on one or multiple sequence similarity parameters. In addition, searches can be restricted according to the predicted subcellular localization of the protein, the DNA strand of the corresponding gene and/or the description of the protein. Massive data search and/or retrieval are available, and different ways of exporting the result are offered. GenoMycDB provides an on-line resource for the functional classification of mycobacterial proteins as well as for the analysis of genome structure, organization, and evolution.

Evolution and Diversity in Human Herpes Simplex Virus Genomes

PubMed Central

Gatherer, Derek; Ochoa, Alejandro; Greenbaum, Benjamin; Dolan, Aidan; Bowden, Rory J.; Enquist, Lynn W.; Legendre, Matthieu; Davison, Andrew J.

2014-01-01

Herpes simplex virus 1 (HSV-1) causes a chronic, lifelong infection in >60% of adults. Multiple recent vaccine trials have failed, with viral diversity likely contributing to these failures. To understand HSV-1 diversity better, we comprehensively compared 20 newly sequenced viral genomes from China, Japan, Kenya, and South Korea with six previously sequenced genomes from the United States, Europe, and Japan. In this diverse collection of passaged strains, we found that one-fifth of the newly sequenced members share a gene deletion and one-third exhibit homopolymeric frameshift mutations (HFMs). Individual strains exhibit genotypic and potential phenotypic variation via HFMs, deletions, short sequence repeats, and single-nucleotide polymorphisms, although the protein sequence identity between strains exceeds 90% on average. In the first genome-scale analysis of positive selection in HSV-1, we found signs of selection in specific proteins and residues, including the fusion protein glycoprotein H. We also confirmed previous results suggesting that recombination has occurred with high frequency throughout the HSV-1 genome. Despite this, the HSV-1 strains analyzed clustered by geographic origin during whole-genome distance analysis. These data shed light on likely routes of HSV-1 adaptation to changing environments and will aid in the selection of vaccine antigens that are invariant worldwide. PMID:24227835

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

PubMed Central

Christensen, Signe; Horowitz, Scott; Bardwell, James C.A.; Olsen, Johan G.; Willemoës, Martin; Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper; Hamelryck, Thomas; Winther, Jakob R.

2017-01-01

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations. PMID:27659562

Order within disorder: Aggrecan chondroitin sulphate-attachment region provides new structural insights into protein sequences classified as disordered

PubMed Central

Jowitt, Thomas A; Murdoch, Alan D; Baldock, Clair; Berry, Richard; Day, Joanna M; Hardingham, Timothy E

2010-01-01

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)-attachment region of human aggrecan (CS-peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS-peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS-peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small-angle X-ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2-G3 construct. The semiordered structure identified in CS-peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins. Proteins 2010. © 2010 Wiley-Liss, Inc. PMID:20806220

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template.

PubMed

Johansson, Kristoffer E; Tidemand Johansen, Nicolai; Christensen, Signe; Horowitz, Scott; Bardwell, James C A; Olsen, Johan G; Willemoës, Martin; Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper; Hamelryck, Thomas; Winther, Jakob R

2016-10-23

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray.

PubMed

Loebel, Madlen; Eckey, Maren; Sotzny, Franziska; Hahn, Elisabeth; Bauer, Sandra; Grabowski, Patricia; Zerweck, Johannes; Holenya, Pavlo; Hanitsch, Leif G; Wittke, Kirsten; Borchmann, Peter; Rüffer, Jens-Ulrich; Hiepe, Falk; Ruprecht, Klemens; Behrends, Uta; Meindl, Carola; Volk, Hans-Dieter; Reimer, Ulf; Scheibenbogen, Carmen

2017-01-01

Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

Genetic characterization of Measles Viruses in China, 2004

PubMed Central

Zhang, Yan; Ji, Yixin; Jiang, Xiaohong; Xu, Songtao; Zhu, Zhen; Zheng, Lei; He, Jilan; Ling, Hua; Wang, Yan; Liu, Yang; Du, Wen; Yang, Xuelei; Mao, Naiying; Xu, Wenbo

2008-01-01

Genetic characterization of wild-type measles virus was studied using nucleotide sequencing of the C-terminal region of the N protein gene and phylogenetic analysis on 59 isolates from 16 provinces of China in 2004. The results showed that all of the isolates belonged to genotype H1. 51 isolates were belonged to cluster 1 and 8 isolates were cluster 2 and Viruses from both clusters were distributed throughout China without distinct geographic pattern. The nucleotide sequence and predicted amino acid homologies of the 59 H1 strains were 96.5%–100% and 95.7%–100%, respectively. The report showed that the transmission pattern of genotype H1 viruses in China in 2004 was consistent with ongoing endemic transmission of multiple lineages of a single, endemic genotype. Multiple transmission pathways leaded to multiple lineages within endemic genotype. PMID:18928575

Automated use of mutagenesis data in structure prediction.

PubMed

Nanda, Vikas; DeGrado, William F

2005-05-15

In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods. Copyright 2005 Wiley-Liss, Inc.

Sequence evidence for the symbiotic origins of chloroplasts and mitochondria

NASA Technical Reports Server (NTRS)

George, D. G.; Hunt, L. T.; Dayhoff, M. O.

1983-01-01

The origin of mitochondria and chloroplasts is investigated on the basis of prokaryotic and early-eukaryotic evolutionary trees derived from protein and nucleic-acid sequences by the method of Dayhoff (1979). Trees for bacterial ferrodoxins, 5S ribosomal RNA, c-type cytochromes, the lipid-binding subunit of ATPase, and dihydrofolate reductase are presented and discussed. Good agreement among the trees is found, and it is argued that the mitochondria and chloroplasts evolved by multiple symbiotic events.

GeneBee-net: Internet-based server for analyzing biopolymers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brodsky, L.I.; Ivanov, V.V.; Nikolaev, V.K.

This work describes a network server for searching databanks of biopolymer structures and performing other biocomputing procedures; it is available via direct Internet connection. Basic server procedures are dedicated to homology (similarity) search of sequence and 3D structure of proteins. The homologies found could be used to build multiple alignments, predict protein and RNA secondary structure, and construct phylogenetic trees. In addition to traditional methods of sequence similarity search, the authors propose {open_quotes}non-matrix{close_quotes} (correlational) search. An analogous approach is used to identify regions of similar tertiary structure of proteins. Algorithm concepts and usage examples are presented for new methods. Servicemore » logic is based upon interaction of a client program and server procedures. The client program allows the compilation of queries and the processing of results of an analysis.« less

SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments.

PubMed

Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

2013-07-01

Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype-phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying 'hot' or 'cold' regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype-phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/.

Application of the MIDAS approach for analysis of lysine acetylation sites.

PubMed

Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

2013-01-01

Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation.

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

PubMed

Tomita, Toshio; Mizumachi, Yoshihiro; Chong, Kang; Ogawa, Kanako; Konishi, Norihide; Sugawara-Tomita, Noriko; Dohmae, Naoshi; Hashimoto, Yohichi; Takio, Koji

2004-12-24

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Deanne W. Sammond | NREL

Science.gov Websites

Interactions," Journal of Biomolecular Structure & Dynamics (2009) "Structure-Based Protocol for from left to right with several dots of multiple colors. "Cellulase Linkers Are Optimized Based on the Sequence and Structure of a Protein-Binding Peptide," Journal of the American Chemical

Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation.

PubMed

Vedantam, Gayatri; Knopf, Sarah; Hecht, David W

2006-01-01

Tn5520 is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistant Bacteroides fragilis clinical isolate. When a conjugation apparatus is provided in trans, Tn5520 is mobilized (transferred) efficiently within, and from, both Bacteroides spp. and Escherichia coli. Only two genes are present on Tn5520; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn5520 mobility. The focus of this study was to identify the Tn5520 origin of conjugative transfer (oriT) and to study BmpH-oriT binding. We delimited the functional Tn5520 oriT to a 71 bp sequence upstream of the bmpH gene. A plasmid vector harbouring this minimal 71 bp oriT was mobilized at the same frequency as that of intact Tn5520. The minimal oriT contains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5'-GCTAC-3') was also identified within the minimal oriT; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn5520 oriT fragment in electrophoretic mobility shift assays. We also identified non-nick site sequences within the minimal oriT that were essential for mobilization. We hypothesize that transposon-based single Mob protein systems may contribute to efficient gene dissemination from Bacteroides spp., because fewer DNA processing proteins are required for relaxosome formation.

Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR

PubMed Central

Cutrale, Francesco; Salih, Anya; Gratton, Enrico

2013-01-01

The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein. PMID:24040513

An efficient, versatile and scalable pattern growth approach to mine frequent patterns in unaligned protein sequences.

PubMed

Ye, Kai; Kosters, Walter A; Ijzerman, Adriaan P

2007-03-15

Pattern discovery in protein sequences is often based on multiple sequence alignments (MSA). The procedure can be computationally intensive and often requires manual adjustment, which may be particularly difficult for a set of deviating sequences. In contrast, two algorithms, PRATT2 (http//www.ebi.ac.uk/pratt/) and TEIRESIAS (http://cbcsrv.watson.ibm.com/) are used to directly identify frequent patterns from unaligned biological sequences without an attempt to align them. Here we propose a new algorithm with more efficiency and more functionality than both PRATT2 and TEIRESIAS, and discuss some of its applications to G protein-coupled receptors, a protein family of important drug targets. In this study, we designed and implemented six algorithms to mine three different pattern types from either one or two datasets using a pattern growth approach. We compared our approach to PRATT2 and TEIRESIAS in efficiency, completeness and the diversity of pattern types. Compared to PRATT2, our approach is faster, capable of processing large datasets and able to identify the so-called type III patterns. Our approach is comparable to TEIRESIAS in the discovery of the so-called type I patterns but has additional functionality such as mining the so-called type II and type III patterns and finding discriminating patterns between two datasets. The source code for pattern growth algorithms and their pseudo-code are available at http://www.liacs.nl/home/kosters/pg/.

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, P.J.; Walthers, E.A.; Richmond, K.L.

1997-04-01

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five Polymorphisms differed by the presence Of two to six copies of the 12-bp tandem repeat 5{prime}-CAATATCAACAA-3{prime}. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats aremore » generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations. 22 refs., 4 figs., 3 tabs.« less

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites.

PubMed

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-04-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass ( Zostera marina ) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae . They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites

PubMed Central

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-01-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses. PMID:29628822

Non-redundant patent sequence databases with value-added annotations at two levels

PubMed Central

Li, Weizhong; McWilliam, Hamish; de la Torre, Ana Richart; Grodowski, Adam; Benediktovich, Irina; Goujon, Mickael; Nauche, Stephane; Lopez, Rodrigo

2010-01-01

The European Bioinformatics Institute (EMBL-EBI) provides public access to patent data, including abstracts, chemical compounds and sequences. Sequences can appear multiple times due to the filing of the same invention with multiple patent offices, or the use of the same sequence by different inventors in different contexts. Information relating to the source invention may be incomplete, and biological information available in patent documents elsewhere may not be reflected in the annotation of the sequence. Search and analysis of these data have become increasingly challenging for both the scientific and intellectual-property communities. Here, we report a collection of non-redundant patent sequence databases, which cover the EMBL-Bank nucleotides patent class and the patent protein databases and contain value-added annotations from patent documents. The databases were created at two levels by the use of sequence MD5 checksums. Sequences within a level-1 cluster are 100% identical over their whole length. Level-2 clusters were defined by sub-grouping level-1 clusters based on patent family information. Value-added annotations, such as publication number corrections, earliest publication dates and feature collations, significantly enhance the quality of the data, allowing for better tracking and cross-referencing. The databases are available format: http://www.ebi.ac.uk/patentdata/nr/. PMID:19884134

Non-redundant patent sequence databases with value-added annotations at two levels.

PubMed

Li, Weizhong; McWilliam, Hamish; de la Torre, Ana Richart; Grodowski, Adam; Benediktovich, Irina; Goujon, Mickael; Nauche, Stephane; Lopez, Rodrigo

2010-01-01

The European Bioinformatics Institute (EMBL-EBI) provides public access to patent data, including abstracts, chemical compounds and sequences. Sequences can appear multiple times due to the filing of the same invention with multiple patent offices, or the use of the same sequence by different inventors in different contexts. Information relating to the source invention may be incomplete, and biological information available in patent documents elsewhere may not be reflected in the annotation of the sequence. Search and analysis of these data have become increasingly challenging for both the scientific and intellectual-property communities. Here, we report a collection of non-redundant patent sequence databases, which cover the EMBL-Bank nucleotides patent class and the patent protein databases and contain value-added annotations from patent documents. The databases were created at two levels by the use of sequence MD5 checksums. Sequences within a level-1 cluster are 100% identical over their whole length. Level-2 clusters were defined by sub-grouping level-1 clusters based on patent family information. Value-added annotations, such as publication number corrections, earliest publication dates and feature collations, significantly enhance the quality of the data, allowing for better tracking and cross-referencing. The databases are available format: http://www.ebi.ac.uk/patentdata/nr/.

Evolution-Based Functional Decomposition of Proteins

PubMed Central

Rivoire, Olivier; Reynolds, Kimberly A.; Ranganathan, Rama

2016-01-01

The essential biological properties of proteins—folding, biochemical activities, and the capacity to adapt—arise from the global pattern of interactions between amino acid residues. The statistical coupling analysis (SCA) is an approach to defining this pattern that involves the study of amino acid coevolution in an ensemble of sequences comprising a protein family. This approach indicates a functional architecture within proteins in which the basic units are coupled networks of amino acids termed sectors. This evolution-based decomposition has potential for new understandings of the structural basis for protein function. To facilitate its usage, we present here the principles and practice of the SCA and introduce new methods for sector analysis in a python-based software package (pySCA). We show that the pattern of amino acid interactions within sectors is linked to the divergence of functional lineages in a multiple sequence alignment—a model for how sector properties might be differentially tuned in members of a protein family. This work provides new tools for studying proteins and for generally testing the concept of sectors as the principal units of function and adaptive variation. PMID:27254668

Tenebrio molitor antifreeze protein gene identification and regulation.

PubMed

Qin, Wensheng; Walker, Virginia K

2006-02-15

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Characterization of Proteoforms with Unknown Post-translational Modifications Using the MIScore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kou, Qiang; Zhu, Binhai; Wu, Si

Various proteoforms may be generated from a single gene due to primary structure alterations (PSAs) such as genetic variations, alternative splicing, and post-translational modifications (PTMs). Top-down mass spectrometry is capable of analyzing intact proteins and identifying patterns of multiple PSAs, making it the method of choice for studying complex proteoforms. In top-down proteomics, proteoform identification is often performed by searching tandem mass spectra against a protein sequence database that contains only one reference protein sequence for each gene or transcript variant in a proteome. Because of the incompleteness of the protein database, an identified proteoform may contain unknown PSAs comparedmore » with the reference sequence. Proteoform characterization is to identify and localize PSAs in a proteoform. Although many software tools have been proposed for proteoform identification by top-down mass spectrometry, the characterization of proteoforms in identified proteoform-spectrum matches still relies mainly on manual annotation. We propose to use the Modification Identification Score (MIScore), which is based on Bayesian models, to automatically identify and localize PTMs in proteoforms. Experiments showed that the MIScore is accurate in identifying and localizing one or two modifications.« less

MetaPSICOV: combining coevolution methods for accurate prediction of contacts and long range hydrogen bonding in proteins.

PubMed

Jones, David T; Singh, Tanya; Kosciolek, Tomasz; Tetchner, Stuart

2015-04-01

Recent developments of statistical techniques to infer direct evolutionary couplings between residue pairs have rendered covariation-based contact prediction a viable means for accurate 3D modelling of proteins, with no information other than the sequence required. To extend the usefulness of contact prediction, we have designed a new meta-predictor (MetaPSICOV) which combines three distinct approaches for inferring covariation signals from multiple sequence alignments, considers a broad range of other sequence-derived features and, uniquely, a range of metrics which describe both the local and global quality of the input multiple sequence alignment. Finally, we use a two-stage predictor, where the second stage filters the output of the first stage. This two-stage predictor is additionally evaluated on its ability to accurately predict the long range network of hydrogen bonds, including correctly assigning the donor and acceptor residues. Using the original PSICOV benchmark set of 150 protein families, MetaPSICOV achieves a mean precision of 0.54 for top-L predicted long range contacts-around 60% higher than PSICOV, and around 40% better than CCMpred. In de novo protein structure prediction using FRAGFOLD, MetaPSICOV is able to improve the TM-scores of models by a median of 0.05 compared with PSICOV. Lastly, for predicting long range hydrogen bonding, MetaPSICOV-HB achieves a precision of 0.69 for the top-L/10 hydrogen bonds compared with just 0.26 for the baseline MetaPSICOV. MetaPSICOV is available as a freely available web server at http://bioinf.cs.ucl.ac.uk/MetaPSICOV. Raw data (predicted contact lists and 3D models) and source code can be downloaded from http://bioinf.cs.ucl.ac.uk/downloads/MetaPSICOV. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

RNA editing: trypanosomes rewrite the genetic code.

PubMed

Stuart, K

1998-01-01

The understanding of how genetic information is stored and expressed has advanced considerably since the "central dogma" asserted that genetic information flows from the nucleotide sequence of DNA to that of messenger RNA (mRNA) which in turn specifies the amino acid sequence of a protein. It was found that genetic information can be stored as RNA (e.g. in RNA viruses) and can flow from RNA to DNA by reverse transcriptase enzyme activity. In addition, some genes contain introns, nucleotide sequences that are removed from their RNA (by RNA splicing) and thus are not represented in the resultant protein. Furthermore, alternative splicing was found to produce variant proteins from a single gene. More recently, the study of trypanosome parasites revealed an unexpected and indeed counter-intuitive genetic complexity. Genetic information for a single protein can be dispersed among several (DNA) genes in these organisms. One of these genes specifies an encrypted precursor mRNA that is converted to a functional mRNA by a process called RNA editing that inserts and deletes uridylate nucleotides. The sequence of the edited mRNA is specified by multiple small RNAs, named guide RNAs, (gRNAs) each of which is encoded in a separate gene. Thus, edited mRNA sequences are assembled from multiple genes by the transfer of information from one type of RNA to another. The existence of editing was surprising but has stimulated the discovery of other types of RNA editing. The Stuart laboratory has been exploring RNA editing in trypanosomes from the time of its discovery. They found dramatic differences between the mitochondrial gene sequences and those of the corresponding mRNAs, which indicated editing by the insertion and deletion of uridylates. Some editing was modest; simply eliminating shifts in sequence register of minimally extending the protein coding sequence. However, editing of many mRNAs was startingly extensive. The RNA sequence was essentially entirely remodeled with its sequence more the result of editing than the gene sequence. The identities of genes for such extensively edited RNA were not recognizable from the DNA sequence but they were readily identifiable from the edited mRNA sequence. Thus, despite the complex and extensive editing the resultant mRNA sequence is precise. Characterization of partially edited RNAs indicated that editing proceeds in the direction opposite to that used to specify the protein which reflects the use of the gRNAs. The numerous gRNAs that are used for editing are encoded in the DNA molecules whose role was previously a mystery. Using information gained in our earlier studies, the Stuart group developed an in vitro system that reproduces the fundamental process of editing in order to resolve the mechanism by which it occurs. They determined that editing entails a series of enzymatic steps rather than the mechanism used in RNA splicing. They also showed that chimeric gRNA-mRNA molecules are aberrant by-products of editing rather than intermediates in the process as had been proposed. Additional studies are exploring precisely how the number of added and deleted uridylates is specified by the gRNA. The Stuart laboratory showed that editing is performed by an aggregation of enzymes that catalyze the separate steps of editing. It also developed a method to purify this multimolecule complex that contains several, perhaps tens of, proteins. This will allow the study of its composition and the functions of its component parts. Indeed, the gene for one component has been identified and its detailed characterization begun. These studies are developing tools to explore related processes. An early finding in the lab was that the various mRNAs are differentially edited during the life cycle of the parasite. The pattern of this editing indicates that editing serves to regulate the alternation between two modes of energy generation. This regulation is coordinated with other events that are occurring during the life c

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Resolution of Site-Specific Conformational Heterogeneity in Proline-Rich Molecular Recognition by Src Homology 3 Domains.

PubMed

Horness, Rachel E; Basom, Edward J; Mayer, John P; Thielges, Megan C

2016-02-03

Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C-D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C-D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C-D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C-D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with (13)C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.

New Insights from Elucidating the Role of LMP1 in Nasopharyngeal Carcinoma

PubMed Central

Shair, Kathy H. Y.; Reddy, Akhil

2018-01-01

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) oncogenic protein that has no intrinsic enzymatic activity or sequence homology to cellular or viral proteins. The oncogenic potential of LMP1 has been ascribed to pleiotropic signaling properties initiated through protein-protein interactions in cytosolic membrane compartments, but the effects of LMP1 extend to nuclear and extracellular processes. Although LMP1 is one of the latent genes required for EBV-immortalization of B cells, the biology of LMP1 in the pathogenesis of the epithelial cancer nasopharyngeal carcinoma (NPC) is more complex. NPC is prevalent in specific regions of the world with high incidence in southeast China. The epidemiology and time interval from seroconversion to NPC onset in adults would suggest the involvement of multiple risk factors that complement the establishment of a latent and persistent EBV infection. The contribution of LMP1 to EBV pathogenesis in polarized epithelia has only recently begun to be elucidated. Furthermore, the LMP1 gene has emerged as one of the most divergent sequences in the EBV genome. This review will discuss the significance of recent advances in NPC research from elucidating LMP1 function in epithelial cells and lessons that could be learned from mining LMP1 sequence diversity. PMID:29561768

Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

PubMed

Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-04-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

Sequence and structural characterization of Trx-Grx type of monothiol glutaredoxins from Ashbya gossypii.

PubMed

Yadav, Saurabh; Kumari, Pragati; Kushwaha, Hemant Ritturaj

2013-01-01

Glutaredoxins are enzymatic antioxidants which are small, ubiquitous, glutathione dependent and essentially classified under thioredoxin-fold superfamily. Glutaredoxins are classified into two types: dithiol and monothiol. Monothiol glutaredoxins which carry the signature "CGFS" as a redox active motif is known for its role in oxidative stress, inside the cell. In the present analysis, the 138 amino acid long monothiol glutaredoxin, AgGRX1 from Ashbya gossypii was identified and has been used for the analysis. The multiple sequence alignment of the AgGRX1 protein sequence revealed the characteristic motif of typical monothiol glutaredoxin as observed in various other organisms. The proposed structure of the AgGRX1 protein was used to analyze signature folds related to the thioredoxin superfamily. Further, the study highlighted the structural features pertaining to the complex mechanism of glutathione docking and interacting residues.

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

PubMed

Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

2010-06-01

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

Comparison of the protein-coding gene content of Chlamydia trachomatis and Protochlamydia amoebophila using a Raspberry Pi computer.

PubMed

Robson, James F; Barker, Daniel

2015-10-13

To demonstrate the bioinformatics capabilities of a low-cost computer, the Raspberry Pi, we present a comparison of the protein-coding gene content of two species in phylum Chlamydiae: Chlamydia trachomatis, a common sexually transmitted infection of humans, and Candidatus Protochlamydia amoebophila, a recently discovered amoebal endosymbiont. Identifying species-specific proteins and differences in protein families could provide insights into the unique phenotypes of the two species. Using a Raspberry Pi computer, sequence similarity-based protein families were predicted across the two species, C. trachomatis and P. amoebophila, and their members counted. Examples include nine multi-protein families unique to C. trachomatis, 132 multi-protein families unique to P. amoebophila and one family with multiple copies in both. Most families unique to C. trachomatis were polymorphic outer-membrane proteins. Additionally, multiple protein families lacking functional annotation were found. Predicted functional interactions suggest one of these families is involved with the exodeoxyribonuclease V complex. The Raspberry Pi computer is adequate for a comparative genomics project of this scope. The protein families unique to P. amoebophila may provide a basis for investigating the host-endosymbiont interaction. However, additional species should be included; and further laboratory research is required to identify the functions of unknown or putative proteins. Multiple outer membrane proteins were found in C. trachomatis, suggesting importance for host evasion. The tyrosine transport protein family is shared between both species, with four proteins in C. trachomatis and two in P. amoebophila. Shared protein families could provide a starting point for discovery of wide-spectrum drugs against Chlamydiae.

COACH: profile-profile alignment of protein families using hidden Markov models.

PubMed

Edgar, Robert C; Sjölander, Kimmen

2004-05-22

Alignments of two multiple-sequence alignments, or statistical models of such alignments (profiles), have important applications in computational biology. The increased amount of information in a profile versus a single sequence can lead to more accurate alignments and more sensitive homolog detection in database searches. Several profile-profile alignment methods have been proposed and have been shown to improve sensitivity and alignment quality compared with sequence-sequence methods (such as BLAST) and profile-sequence methods (e.g. PSI-BLAST). Here we present a new approach to profile-profile alignment we call Comparison of Alignments by Constructing Hidden Markov Models (HMMs) (COACH). COACH aligns two multiple sequence alignments by constructing a profile HMM from one alignment and aligning the other to that HMM. We compare the alignment accuracy of COACH with two recently published methods: Yona and Levitt's prof_sim and Sadreyev and Grishin's COMPASS. On two sets of reference alignments selected from the FSSP database, we find that COACH is able, on average, to produce alignments giving the best coverage or the fewest errors, depending on the chosen parameter settings. COACH is freely available from www.drive5.com/lobster

Multiple layers of temporal and spatial control regulate accumulation of the fruiting body-specific protein APP in Sordaria macrospora and Neurospora crassa.

PubMed

Nowrousian, Minou; Piotrowski, Markus; Kück, Ulrich

2007-07-01

During fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. We have isolated a protein from the ascomycete Sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. The protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant perithecial protein). app transcript occurs only after the onset of sexual development; however, the formation of ascospores is not a prerequisite for APP accumulation. The transcript of the Neurospora crassa ortholog is present prior to fertilization, but the protein accumulates only after fertilization. In crosses of N. crassa Deltaapp strains with the wild type, APP accumulates when the wild type serves as female parent, but not in the reciprocal cross; thus, the presence of a functional female app allele is necessary and sufficient for APP accumulation. These findings highlight multiple layers of temporal and spatial control of gene expression during fungal development.

Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

PubMed Central

O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

2015-01-01

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

AlexSys: a knowledge-based expert system for multiple sequence alignment construction and analysis

PubMed Central

Aniba, Mohamed Radhouene; Poch, Olivier; Marchler-Bauer, Aron; Thompson, Julie Dawn

2010-01-01

Multiple sequence alignment (MSA) is a cornerstone of modern molecular biology and represents a unique means of investigating the patterns of conservation and diversity in complex biological systems. Many different algorithms have been developed to construct MSAs, but previous studies have shown that no single aligner consistently outperforms the rest. This has led to the development of a number of ‘meta-methods’ that systematically run several aligners and merge the output into one single solution. Although these methods generally produce more accurate alignments, they are inefficient because all the aligners need to be run first and the choice of the best solution is made a posteriori. Here, we describe the development of a new expert system, AlexSys, for the multiple alignment of protein sequences. AlexSys incorporates an intelligent inference engine to automatically select an appropriate aligner a priori, depending only on the nature of the input sequences. The inference engine was trained on a large set of reference multiple alignments, using a novel machine learning approach. Applying AlexSys to a test set of 178 alignments, we show that the expert system represents a good compromise between alignment quality and running time, making it suitable for high throughput projects. AlexSys is freely available from http://alnitak.u-strasbg.fr/∼aniba/alexsys. PMID:20530533

Exploring the repeat protein universe through computational protein design

DOE PAGES

Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

2015-12-16

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less

Prediction of beta-turns and beta-turn types by a novel bidirectional Elman-type recurrent neural network with multiple output layers (MOLEBRNN).

PubMed

Kirschner, Andreas; Frishman, Dmitrij

2008-10-01

Prediction of beta-turns from amino acid sequences has long been recognized as an important problem in structural bioinformatics due to their frequent occurrence as well as their structural and functional significance. Because various structural features of proteins are intercorrelated, secondary structure information has been often employed as an additional input for machine learning algorithms while predicting beta-turns. Here we present a novel bidirectional Elman-type recurrent neural network with multiple output layers (MOLEBRNN) capable of predicting multiple mutually dependent structural motifs and demonstrate its efficiency in recognizing three aspects of protein structure: beta-turns, beta-turn types, and secondary structure. The advantage of our method compared to other predictors is that it does not require any external input except for sequence profiles because interdependencies between different structural features are taken into account implicitly during the learning process. In a sevenfold cross-validation experiment on a standard test dataset our method exhibits the total prediction accuracy of 77.9% and the Mathew's Correlation Coefficient of 0.45, the highest performance reported so far. It also outperforms other known methods in delineating individual turn types. We demonstrate how simultaneous prediction of multiple targets influences prediction performance on single targets. The MOLEBRNN presented here is a generic method applicable in a variety of research fields where multiple mutually depending target classes need to be predicted. http://webclu.bio.wzw.tum.de/predator-web/.

Evidence for the Concerted Evolution between Short Linear Protein Motifs and Their Flanking Regions

PubMed Central

Chica, Claudia; Diella, Francesca; Gibson, Toby J.

2009-01-01

Background Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein–protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids. Results The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co–evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif–mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions. Conclusion The results suggest that flanking regions are relevant for linear motif–mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise. PMID:19584925

Diversity of the luciferin binding protein gene in bioluminescent dinoflagellates--insights from a new gene in Noctiluca scintillans and sequences from gonyaulacoid genera.

PubMed

Valiadi, Martha; Iglesias-Rodriguez, Maria Debora

2014-01-01

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re-organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within-strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

Characterisation of a large family of polymorphic collagen-like proteins in the endospore-forming bacterium Pasteuria ramosa.

PubMed

McElroy, Kerensa; Mouton, Laurence; Du Pasquier, Louis; Qi, Weihong; Ebert, Dieter

2011-09-01

Collagen-like proteins containing glycine-X-Y repeats have been identified in several pathogenic bacteria potentially involved in virulence. Recently, a collagen-like surface protein, Pcl1a, was identified in Pasteuria ramosa, a spore-forming parasite of Daphnia. Here we characterise 37 novel putative P. ramosa collagen-like protein genes (PCLs). PCR amplification and sequencing across 10 P. ramosa strains showed they were polymorphic, distinguishing genotypes matching known differences in Daphnia/P. ramosa interaction specificity. Thirty PCLs could be divided into four groups based on sequence similarity, conserved N- and C-terminal regions and G-X-Y repeat structure. Group 1, Group 2 and Group 3 PCLs formed triplets within the genome, with one member from each group represented in each triplet. Maximum-likelihood trees suggested that these groups arose through multiple instances of triplet duplication. For Group 1, 2, 3 and 4 PCLs, X was typically proline and Y typically threonine, consistent with other bacterial collagen-like proteins. The amino acid composition of Pcl2 closely resembled Pcl1a, with X typically being glutamic acid or aspartic acid and Y typically being lysine or glutamine. Pcl2 also showed sequence similarity to Pcl1a and contained a predicted signal peptide, cleavage site and transmembrane domain, suggesting that it is a surface protein. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

PubMed

Ma, Xingliang; Liu, Yao-Guang

2016-07-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Identification and Analysis of Novel Amino-Acid Sequence Repeats in Bacillus anthracis str. Ames Proteome Using Computational Tools

PubMed Central

Hemalatha, G. R.; Rao, D. Satyanarayana; Guruprasad, L.

2007-01-01

We have identified four repeats and ten domains that are novel in proteins encoded by the Bacillus anthracis str. Ames proteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 57-amino-acid-residue PxV domain, (2) 122-amino-acid-residue FxF domain, (3) 111-amino-acid-residue YEFF domain, (4) 109-amino-acid-residue IMxxH domain, (5) 103-amino-acid-residue VxxT domain, (6) 84-amino-acid-residue ExW domain, (7) 104-amino-acid-residue NTGFIG domain, (8) 36-amino-acid-residue NxGK repeat, (9) 95-amino-acid-residue VYV domain, (10) 75-amino-acid-residue KEWE domain, (11) 59-amino-acid-residue AFL domain, (12) 53-amino-acid-residue RIDVK repeat, (13) (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure. PMID:17538688

Accurate Simulation and Detection of Coevolution Signals in Multiple Sequence Alignments

PubMed Central

Ackerman, Sharon H.; Tillier, Elisabeth R.; Gatti, Domenico L.

2012-01-01

Background While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones. Methodology/Principal Findings We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the in silico evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs. Conclusions/Significance Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most effective in each case. PMID:23091608

The unfolding mechanism of monomeric mutant SOD1 by simulated force spectroscopy.

PubMed

Habibi, Mona; Rottler, Jörg; Plotkin, Steven S

2017-11-01

Mechanical unfolding of mutated apo, disulfide-reduced, monomeric superoxide dismutase 1 protein (SOD1) has been simulated via force spectroscopy techniques, using both an all-atom (AA), explicit solvent model and a coarse-grained heavy-atom Gō (HA-Gō) model. The HA-Gō model was implemented at two different pulling speeds for comparison. The most-common sequence of unfolding in the AA model agrees well with the most-common unfolding sequence of the HA-Gō model, when the same normalized pulling rate was used. Clustering of partially-native structures as the protein unfolds shows that the AA and HA-Gō models both exhibit a dominant pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches after the protein is about half-unfolded. The force-extension curve exhibits multiple force drops, which are concomitant with jumps in the local interaction potential energy between specific β-strands in the protein. These sudden jumps in the potential energy coincide with the dissociation of specific pairs of β-strands, and thus intermediate unfolding events. The most common sequence of β-strand dissociation in the unfolding pathway of the AA model is β-strands 5, 4, 8, 7, 1, 2, then finally β-strands 3 and 6. The observation that β-strand 5 is among the first to unfold here, but the last to unfold in simulations of loop-truncated SOD1, could imply the existence of an evolutionary compensation mechanism, which would stabilize β-strands flanking long loops against their entropic penalty by strengthening intramolecular interactions. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Harnessing Omics Big Data in Nine Vertebrate Species by Genome-Wide Prioritization of Sequence Variants with the Highest Predicted Deleterious Effect on Protein Function.

PubMed

Rozman, Vita; Kunej, Tanja

2018-05-10

Harnessing the genomics big data requires innovation in how we extract and interpret biologically relevant variants. Currently, there is no established catalog of prioritized missense variants associated with deleterious protein function phenotypes. We report in this study, to the best of our knowledge, the first genome-wide prioritization of sequence variants with the most deleterious effect on protein function (potentially deleterious variants [pDelVars]) in nine vertebrate species: human, cattle, horse, sheep, pig, dog, rat, mouse, and zebrafish. The analysis was conducted using the Ensembl/BioMart tool. Genes comprising pDelVars in the highest number of examined species were identified using a Python script. Multiple genomic alignments of the selected genes were built to identify interspecies orthologous potentially deleterious variants, which we defined as the "ortho-pDelVars." Genome-wide prioritization revealed that in humans, 0.12% of the known variants are predicted to be deleterious. In seven out of nine examined vertebrate species, the genes encoding the multiple PDZ domain crumbs cell polarity complex component (MPDZ) and the transforming acidic coiled-coil containing protein 2 (TACC2) comprise pDelVars. Five interspecies ortho-pDelVars were identified in three genes. These findings offer new ways to harness genomics big data by facilitating the identification of functional polymorphisms in humans and animal models and thus provide a future basis for optimization of protocols for whole genome prioritization of pDelVars and screening of orthologous sequence variants. The approach presented here can inform various postgenomic applications such as personalized medicine and multiomics study of health interventions (iatromics).

Identification and analysis of mutational hotspots in oncogenes and tumour suppressors.

PubMed

Baeissa, Hanadi; Benstead-Hume, Graeme; Richardson, Christopher J; Pearl, Frances M G

2017-03-28

The key to interpreting the contribution of a disease-associated mutation in the development and progression of cancer is an understanding of the consequences of that mutation both on the function of the affected protein and on the pathways in which that protein is involved. Protein domains encapsulate function and position-specific domain based analysis of mutations have been shown to help elucidate their phenotypes. In this paper we examine the domain biases in oncogenes and tumour suppressors, and find that their domain compositions substantially differ. Using data from over 30 different cancers from whole-exome sequencing cancer genomic projects we mapped over one million mutations to their respective Pfam domains to identify which domains are enriched in any of three different classes of mutation; missense, indels or truncations. Next, we identified the mutational hotspots within domain families by mapping small mutations to equivalent positions in multiple sequence alignments of protein domainsWe find that gain of function mutations from oncogenes and loss of function mutations from tumour suppressors are normally found in different domain families and when observed in the same domain families, hotspot mutations are located at different positions within the multiple sequence alignment of the domain. By considering hotspots in tumour suppressors and oncogenes independently, we find that there are different specific positions within domain families that are particularly suited to accommodate either a loss or a gain of function mutation. The position is also dependent on the class of mutation.We find rare mutations co-located with well-known functional mutation hotspots, in members of homologous domain superfamilies, and we detect novel mutation hotspots in domain families previously unconnected with cancer. The results of this analysis can be accessed through the MOKCa database (http://strubiol.icr.ac.uk/extra/MOKCa).

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders.

PubMed

Vienneau-Hathaway, Jannelle M; Brassfield, Elizabeth R; Lane, Amanda Kelly; Collin, Matthew A; Correa-Garhwal, Sandra M; Clarke, Thomas H; Schwager, Evelyn E; Garb, Jessica E; Hayashi, Cheryl Y; Ayoub, Nadia A

2017-03-14

Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Hexagonal ice in pure water and biological NMR samples.

PubMed

Bauer, Thomas; Gath, Julia; Hunkeler, Andreas; Ernst, Matthias; Böckmann, Anja; Meier, Beat H

2017-01-01

Ice, in addition to "liquid" water and protein, is an important component of protein samples for NMR spectroscopy at subfreezing temperatures but it has rarely been observed spectroscopically in this context. We characterize its spectroscopic behavior in the temperature range from 100 to 273 K, and find that it behaves like pure water ice. The interference of magic-angle spinning (MAS) as well as rf multiple-pulse sequences with Bjerrum-defect motion greatly influences the ice spectra.

GWIPS‐viz as a tool for exploring ribosome profiling evidence supporting the synthesis of alternative proteoforms

PubMed Central

Michel, Audrey M.; Ahern, Anna M.; Donohue, Claire A.

2015-01-01

The boundaries of protein coding sequences are more difficult to define at the 5′ end than at the 3′ end due to potential multiple translation initiation sites (TISs). Even in the presence of phylogenetic data, the use of sequence information only may not be sufficient for the accurate identification of TISs. Traditional proteomics approaches may also fail because the N‐termini of newly synthesized proteins are often processed. Thus ribosome profiling (ribo‐seq), producing a snapshot of the ribosome distribution across the entire transcriptome, is an attractive experimental technique for the purpose of TIS location exploration. The GWIPS‐viz (Genome Wide Information on Protein Synthesis visualized) browser (http://gwips.ucc.ie) provides free access to the genomic alignments of ribo‐seq data and corresponding mRNA‐seq data along with relevant annotation tracks. In this brief, we illustrate how GWIPS‐viz can be used to explore the ribosome occupancy at the 5′ ends of protein coding genes to assess the activity of AUG and non‐AUG TISs responsible for the synthesis of proteoforms with alternative or heterogeneous N‐termini. The presence of ribo‐seq tracks for various organisms allows for cross‐species comparison of orthologous genes and the availability of datasets from multiple laboratories permits the assessment of the technical reproducibility of the ribosome densities. PMID:25736862

Optimizing multiple sequence alignments using a genetic algorithm based on three objectives: structural information, non-gaps percentage and totally conserved columns.

PubMed

Ortuño, Francisco M; Valenzuela, Olga; Rojas, Fernando; Pomares, Hector; Florido, Javier P; Urquiza, Jose M; Rojas, Ignacio

2013-09-01

Multiple sequence alignments (MSAs) are widely used approaches in bioinformatics to carry out other tasks such as structure predictions, biological function analyses or phylogenetic modeling. However, current tools usually provide partially optimal alignments, as each one is focused on specific biological features. Thus, the same set of sequences can produce different alignments, above all when sequences are less similar. Consequently, researchers and biologists do not agree about which is the most suitable way to evaluate MSAs. Recent evaluations tend to use more complex scores including further biological features. Among them, 3D structures are increasingly being used to evaluate alignments. Because structures are more conserved in proteins than sequences, scores with structural information are better suited to evaluate more distant relationships between sequences. The proposed multiobjective algorithm, based on the non-dominated sorting genetic algorithm, aims to jointly optimize three objectives: STRIKE score, non-gaps percentage and totally conserved columns. It was significantly assessed on the BAliBASE benchmark according to the Kruskal-Wallis test (P < 0.01). This algorithm also outperforms other aligners, such as ClustalW, Multiple Sequence Alignment Genetic Algorithm (MSA-GA), PRRP, DIALIGN, Hidden Markov Model Training (HMMT), Pattern-Induced Multi-sequence Alignment (PIMA), MULTIALIGN, Sequence Alignment Genetic Algorithm (SAGA), PILEUP, Rubber Band Technique Genetic Algorithm (RBT-GA) and Vertical Decomposition Genetic Algorithm (VDGA), according to the Wilcoxon signed-rank test (P < 0.05), whereas it shows results not significantly different to 3D-COFFEE (P > 0.05) with the advantage of being able to use less structures. Structural information is included within the objective function to evaluate more accurately the obtained alignments. The source code is available at http://www.ugr.es/~fortuno/MOSAStrE/MO-SAStrE.zip.

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb

2011-10-28

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and themore » cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.« less

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

PubMed Central

Russo, James J.; Bohenzky, Roy A.; Chien, Ming-Cheng; Chen, Jing; Yan, Ming; Maddalena, Dawn; Parry, J. Preston; Peruzzi, Daniela; Edelman, Isidore S.; Chang, Yuan; Moore, Patrick S.

1996-01-01

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. PMID:8962146

Multiple ice-binding proteins of probable prokaryotic origin in an Antarctic lake alga, Chlamydomonas sp. ICE-MDV (Chlorophyceae).

PubMed

Raymond, James A; Morgan-Kiss, Rachael

2017-08-01

Ice-associated algae produce ice-binding proteins (IBPs) to prevent freezing damage. The IBPs of the three chlorophytes that have been examined so far share little similarity across species, making it likely that they were acquired by horizontal gene transfer (HGT). To clarify the importance and source of IBPs in chlorophytes, we sequenced the IBP genes of another Antarctic chlorophyte, Chlamydomonas sp. ICE-MDV (Chlamy-ICE). Genomic DNA and total RNA were sequenced and screened for known ice-associated genes. Chlamy-ICE has as many as 50 IBP isoforms, indicating that they have an important role in survival. The IBPs are of the DUF3494 type and have similar exon structures. The DUF3494 sequences are much more closely related to prokaryotic sequences than they are to sequences in other chlorophytes, and the chlorophyte IBP and ribosomal 18S phylogenies are dissimilar. The multiple IBP isoforms found in Chlamy-ICE and other algae may allow the algae to adapt to a greater variety of ice conditions than prokaryotes, which typically have a single IBP gene. The predicted structure of the DUF3494 domain has an ice-binding face with an orderly array of hydrophilic side chains. The results indicate that Chlamy-ICE acquired its IBP genes by HGT in a single event. The acquisitions of IBP genes by this and other species of Antarctic algae by HGT appear to be key evolutionary events that allowed algae to extend their ranges into polar environments. © 2017 Phycological Society of America.

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

PubMed

Tapia, Lorena I; Shaw, Chad A; Aideyan, Letisha O; Jewell, Alan M; Dawson, Brian C; Haq, Taha R; Piedra, Pedro A

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Gene Sequence Variability of the Three Surface Proteins of Human Respiratory Syncytial Virus (HRSV) in Texas

PubMed Central

Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544

Phylogenetic place of guinea pigs: no support of the rodent-polyphyly hypothesis from maximum-likelihood analyses of multiple protein sequences.

PubMed

Cao, Y; Adachi, J; Yano, T; Hasegawa, M

1994-07-01

Graur et al.'s (1991) hypothesis that the guinea pig-like rodents have an evolutionary origin within mammals that is separate from that of other rodents (the rodent-polyphyly hypothesis) was reexamined by the maximum-likelihood method for protein phylogeny, as well as by the maximum-parsimony and neighbor-joining methods. The overall evidence does not support Graur et al.'s hypothesis, which radically contradicts the traditional view of rodent monophyly. This work demonstrates that we must be careful in choosing a proper method for phylogenetic inference and that an argument based on a small data set (with respect to the length of the sequence and especially the number of species) may be unstable.

Robust one-Tube Ω-PCR Strategy Accelerates Precise Sequence Modification of Plasmids for Functional Genomics

PubMed Central

Chen, Letian; Wang, Fengpin; Wang, Xiaoyu; Liu, Yao-Guang

2013-01-01

Functional genomics requires vector construction for protein expression and functional characterization of target genes; therefore, a simple, flexible and low-cost molecular manipulation strategy will be highly advantageous for genomics approaches. Here, we describe a Ω-PCR strategy that enables multiple types of sequence modification, including precise insertion, deletion and substitution, in any position of a circular plasmid. Ω-PCR is based on an overlap extension site-directed mutagenesis technique, and is named for its characteristic Ω-shaped secondary structure during PCR. Ω-PCR can be performed either in two steps, or in one tube in combination with exonuclease I treatment. These strategies have wide applications for protein engineering, gene function analysis and in vitro gene splicing. PMID:23335613

Characterization of a protein that binds multiple sequences in mammalian type C retrovirus enhancers.

PubMed Central

Sun, W; O'Connell, M; Speck, N A

1993-01-01

Mammalian type C retrovirus enhancer factor 1 (MCREF-1) is a nuclear protein that binds several directly repeated sequences (CNGGN6CNGG) in the Moloney and Friend murine leukemia virus (MLV) enhancers (N. R. Manley, M. O'Connell, W. Sun, N. A. Speck, and N. Hopkins, J. Virol. 67:1967-1975, 1993). In this paper, we describe the partial purification of MCREF-1 from calf thymus nuclei and further characterize the binding properties of MCREF-1. MCREF-1 binds four sites in the Moloney MLV enhancer and three sites in the Friend MLV enhancer. Ethylation interference analysis suggests that the MCREF-1 binding site spans two adjacent minor grooves of DNA. Images PMID:8445719

Fungal proteomics: from identification to function.

PubMed

Doyle, Sean

2011-08-01

Some fungi cause disease in humans and plants, while others have demonstrable potential for the control of insect pests. In addition, fungi are also a rich reservoir of therapeutic metabolites and industrially useful enzymes. Detailed analysis of fungal biochemistry is now enabled by multiple technologies including protein mass spectrometry, genome and transcriptome sequencing and advances in bioinformatics. Yet, the assignment of function to fungal proteins, encoded either by in silico annotated, or unannotated genes, remains problematic. The purpose of this review is to describe the strategies used by many researchers to reveal protein function in fungi, and more importantly, to consolidate the nomenclature of 'unknown function protein' as opposed to 'hypothetical protein' - once any protein has been identified by protein mass spectrometry. A combination of approaches including comparative proteomics, pathogen-induced protein expression and immunoproteomics are outlined, which, when used in combination with a variety of other techniques (e.g. functional genomics, microarray analysis, immunochemical and infection model systems), appear to yield comprehensive and definitive information on protein function in fungi. The relative advantages of proteomic, as opposed to transcriptomic-only, analyses are also described. In the future, combined high-throughput, quantitative proteomics, allied to transcriptomic sequencing, are set to reveal much about protein function in fungi. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites.

PubMed

Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui

2014-06-01

Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted. By initially defining an objective function, the possible optimal degenerate PCR primer profiles could be automatically explored using the heuristic approach of Greedy Best-First-Search. Compared to the previously developed DC-Analyzer, MDC-Analyzer allows for the existence of a small amount of undesired sequences as a tradeoff between the number of degenerate primers and the encoded library size while still providing all the benefits of DC-Analyzer with the ability to randomize multiple contiguous sites. MDC-Analyzer was validated using a series of randomly generated mutation schemes and experimental case studies on the evolution of halohydrin dehalogenase, which proved that the MDC methodology is more efficient than other methods and is particularly well-suited to exploring the sequence space of proteins using data-driven protein engineering strategies.

Parallel algorithms for large-scale biological sequence alignment on Xeon-Phi based clusters.

PubMed

Lan, Haidong; Chan, Yuandong; Xu, Kai; Schmidt, Bertil; Peng, Shaoliang; Liu, Weiguo

2016-07-19

Computing alignments between two or more sequences are common operations frequently performed in computational molecular biology. The continuing growth of biological sequence databases establishes the need for their efficient parallel implementation on modern accelerators. This paper presents new approaches to high performance biological sequence database scanning with the Smith-Waterman algorithm and the first stage of progressive multiple sequence alignment based on the ClustalW heuristic on a Xeon Phi-based compute cluster. Our approach uses a three-level parallelization scheme to take full advantage of the compute power available on this type of architecture; i.e. cluster-level data parallelism, thread-level coarse-grained parallelism, and vector-level fine-grained parallelism. Furthermore, we re-organize the sequence datasets and use Xeon Phi shuffle operations to improve I/O efficiency. Evaluations show that our method achieves a peak overall performance up to 220 GCUPS for scanning real protein sequence databanks on a single node consisting of two Intel E5-2620 CPUs and two Intel Xeon Phi 7110P cards. It also exhibits good scalability in terms of sequence length and size, and number of compute nodes for both database scanning and multiple sequence alignment. Furthermore, the achieved performance is highly competitive in comparison to optimized Xeon Phi and GPU implementations. Our implementation is available at https://github.com/turbo0628/LSDBS-mpi .

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

PubMed

Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

2009-11-20

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.

Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes

PubMed Central

Rodríguez-Bolaños, Monica; Cabrera, Nallely

2016-01-01

The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi, and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei. Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins. PMID:27733588

FIST: a sensory domain for diverse signal transduction pathways in prokaryotes and ubiquitin signaling in eukaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borziak, Kirill; Jouline, Igor B

2007-01-01

Motivation: Sensory domains that are conserved among Bacteria, Archaea and Eucarya are important detectors of common signals detected by living cells. Due to their high sequence divergence, sensory domains are difficult to identify. We systematically look for novel sensory domains using sensitive profile-based searches initi-ated with regions of signal transduction proteins where no known domains can be identified by current domain models. Results: Using profile searches followed by multiple sequence alignment, structure prediction, and domain architecture analysis, we have identified a novel sensory domain termed FIST, which is present in signal transduction proteins from Bacteria, Archaea and Eucarya. Remote similaritymore » to a known ligand-binding fold and chromosomal proximity of FIST-encoding genes to those coding for proteins involved in amino acid metabolism and transport suggest that FIST domains bind small ligands, such as amino acids.« less

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation.

PubMed

Dickman, M B; Ha, Y S; Yang, Z; Adams, B; Huang, C

2003-05-01

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

STING Millennium: a web-based suite of programs for comprehensive and simultaneous analysis of protein structure and sequence

PubMed Central

Neshich, Goran; Togawa, Roberto C.; Mancini, Adauto L.; Kuser, Paula R.; Yamagishi, Michel E. B.; Pappas, Georgios; Torres, Wellington V.; Campos, Tharsis Fonseca e; Ferreira, Leonardo L.; Luna, Fabio M.; Oliveira, Adilton G.; Miura, Ronald T.; Inoue, Marcus K.; Horita, Luiz G.; de Souza, Dimas F.; Dominiquini, Fabiana; Álvaro, Alexandre; Lima, Cleber S.; Ogawa, Fabio O.; Gomes, Gabriel B.; Palandrani, Juliana F.; dos Santos, Gabriela F.; de Freitas, Esther M.; Mattiuz, Amanda R.; Costa, Ivan C.; de Almeida, Celso L.; Souza, Savio; Baudet, Christian; Higa, Roberto H.

2003-01-01

STING Millennium Suite (SMS) is a new web-based suite of programs and databases providing visualization and a complex analysis of molecular sequence and structure for the data deposited at the Protein Data Bank (PDB). SMS operates with a collection of both publicly available data (PDB, HSSP, Prosite) and its own data (contacts, interface contacts, surface accessibility). Biologists find SMS useful because it provides a variety of algorithms and validated data, wrapped-up in a user friendly web interface. Using SMS it is now possible to analyze sequence to structure relationships, the quality of the structure, nature and volume of atomic contacts of intra and inter chain type, relative conservation of amino acids at the specific sequence position based on multiple sequence alignment, indications of folding essential residue (FER) based on the relationship of the residue conservation to the intra-chain contacts and Cα–Cα and Cβ–Cβ distance geometry. Specific emphasis in SMS is given to interface forming residues (IFR)—amino acids that define the interactive portion of the protein surfaces. SMS may simultaneously display and analyze previously superimposed structures. PDB updates trigger SMS updates in a synchronized fashion. SMS is freely accessible for public data at http://www.cbi.cnptia.embrapa.br, http://mirrors.rcsb.org/SMS and http://trantor.bioc.columbia.edu/SMS. PMID:12824333

Identification and Characterization of Multiple Spidroin 1 Genes Encoding Major Ampullate Silk Proteins in Nephila clavipes

PubMed Central

Gaines, William A.; Marcotte, William R.

2010-01-01

Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSp) that consist of a large repeat array flanked by non-repetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We have experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species. PMID:18828837

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

PubMed

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-11-04

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome*

PubMed Central

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-01-01

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR?

PubMed

Illingworth, Christopher J R; Parkes, Kevin E; Snell, Christopher R; Mullineaux, Philip M; Reynolds, Christopher A

2008-03-01

Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

A Protein Domain and Family Based Approach to Rare Variant Association Analysis.

PubMed

Richardson, Tom G; Shihab, Hashem A; Rivas, Manuel A; McCarthy, Mark I; Campbell, Colin; Timpson, Nicholas J; Gaunt, Tom R

2016-01-01

It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals.

Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry

PubMed Central

Zhang, Han; Ge, Ying

2012-01-01

Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research. PMID:22187450

Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.

PubMed

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M; Pedersen, Joel A; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

NASA Astrophysics Data System (ADS)

Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

2012-09-01

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

Study of Binding Interaction between Pif80 Protein Fragment and Aragonite

NASA Astrophysics Data System (ADS)

Du, Yuan-Peng; Chang, Hsun-Hui; Yang, Sheng-Yu; Huang, Shing-Jong; Tsai, Yu-Ju; Huang, Joseph Jen-Tse; Chan, Jerry Chun Chung

2016-08-01

Pif is a crucial protein for the formation of the nacreous layer in Pinctada fucata. Three non-acidic peptide fragments of the aragonite-binding domain (Pif80) are selected, which contain multiple copies of the repeat sequence DDRK, to study the interaction between non-acidic peptides and aragonite. The polypeptides DDRKDDRKGGK (Pif80-11) and DDRKDDRKGGKDDRKDDRKGGK (Pif80-22) have similar binding affinity to aragonite. Solid-state NMR data indicate that the backbones of Pif80-11 and Pif80-22 peptides bound on aragonite adopt a random-coil conformation. Pif80-11 is a lot more effective than Pif80-22 in promoting the nucleation of aragonite on the substrate of β-chitin. Our results suggest that the structural arrangement at a protein-mineral interface depends on the surface structure of the mineral substrate and the protein sequence. The side chains of the basic residues, which function as anchors to the aragonite surface, have uniform structures. The role of basic residues as anchors in protein-mineral interaction may play an important role in biomineralization.

Exome Sequencing Finds a Novel PCSK1 Mutation in a Child With Generalized Malabsorptive Diarrhea and Diabetes Insipidus

PubMed Central

Yourshaw, Michael; Solorzano-Vargas, R. Sergio; Pickett, Lindsay A.; Lindberg, Iris; Wang, Jiafang; Cortina, Galen; Pawlikowska-Haddal, Anna; Baron, Howard; Venick, Robert S.; Nelson, Stanley F.; Martín, Martín G.

2014-01-01

Objectives Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. Methods We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Results Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Conclusions Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations. PMID:24280991

Exome sequencing finds a novel PCSK1 mutation in a child with generalized malabsorptive diarrhea and diabetes insipidus.

PubMed

Yourshaw, Michael; Solorzano-Vargas, R Sergio; Pickett, Lindsay A; Lindberg, Iris; Wang, Jiafang; Cortina, Galen; Pawlikowska-Haddal, Anna; Baron, Howard; Venick, Robert S; Nelson, Stanley F; Martín, Martín G

2013-12-01

Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.

Highly sensitive detection of individual HEAT and ARM repeats with HHpred and COACH.

PubMed

Kippert, Fred; Gerloff, Dietlind L

2009-09-24

HEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat. Testing algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries. A protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high sensitivity at a low false positive rate and will therefore greatly enhance the accuracy of predictions of HEAT and ARM domains.

Highly Sensitive Detection of Individual HEAT and ARM Repeats with HHpred and COACH

PubMed Central

Kippert, Fred; Gerloff, Dietlind L.

2009-01-01

Background HEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat. Methodology and Principal Findings Testing algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries. Significance A protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high sensitivity at a low false positive rate and will therefore greatly enhance the accuracy of predictions of HEAT and ARM domains. PMID:19777061

Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

1988-08-01

The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end ofmore » the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.« less

Rapid search for tertiary fragments reveals protein sequence–structure relationships

PubMed Central

Zhou, Jianfu; Grigoryan, Gevorg

2015-01-01

Finding backbone substructures from the Protein Data Bank that match an arbitrary query structural motif, composed of multiple disjoint segments, is a problem of growing relevance in structure prediction and protein design. Although numerous protein structure search approaches have been proposed, methods that address this specific task without additional restrictions and on practical time scales are generally lacking. Here, we propose a solution, dubbed MASTER, that is both rapid, enabling searches over the Protein Data Bank in a matter of seconds, and provably correct, finding all matches below a user-specified root-mean-square deviation cutoff. We show that despite the potentially exponential time complexity of the problem, running times in practice are modest even for queries with many segments. The ability to explore naturally plausible structural and sequence variations around a given motif has the potential to synthesize its design principles in an automated manner; so we go on to illustrate the utility of MASTER to protein structural biology. We demonstrate its capacity to rapidly establish structure–sequence relationships, uncover the native designability landscapes of tertiary structural motifs, identify structural signatures of binding, and automatically rewire protein topologies. Given the broad utility of protein tertiary fragment searches, we hope that providing MASTER in an open-source format will enable novel advances in understanding, predicting, and designing protein structure. PMID:25420575

MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

PubMed

Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

2012-03-01

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

Photosynthesis Is Widely Distributed among Proteobacteria as Demonstrated by the Phylogeny of PufLM Reaction Center Proteins

PubMed Central

Imhoff, Johannes F.; Rahn, Tanja; Künzel, Sven; Neulinger, Sven C.

2018-01-01

Two different photosystems for performing bacteriochlorophyll-mediated photosynthetic energy conversion are employed in different bacterial phyla. Those bacteria employing a photosystem II type of photosynthetic apparatus include the phototrophic purple bacteria (Proteobacteria), Gemmatimonas and Chloroflexus with their photosynthetic relatives. The proteins of the photosynthetic reaction center PufL and PufM are essential components and are common to all bacteria with a type-II photosynthetic apparatus, including the anaerobic as well as the aerobic phototrophic Proteobacteria. Therefore, PufL and PufM proteins and their genes are perfect tools to evaluate the phylogeny of the photosynthetic apparatus and to study the diversity of the bacteria employing this photosystem in nature. Almost complete pufLM gene sequences and the derived protein sequences from 152 type strains and 45 additional strains of phototrophic Proteobacteria employing photosystem II were compared. The results give interesting and comprehensive insights into the phylogeny of the photosynthetic apparatus and clearly define Chromatiales, Rhodobacterales, Sphingomonadales as major groups distinct from other Alphaproteobacteria, from Betaproteobacteria and from Caulobacterales (Brevundimonas subvibrioides). A special relationship exists between the PufLM sequences of those bacteria employing bacteriochlorophyll b instead of bacteriochlorophyll a. A clear phylogenetic association of aerobic phototrophic purple bacteria to anaerobic purple bacteria according to their PufLM sequences is demonstrated indicating multiple evolutionary lines from anaerobic to aerobic phototrophic purple bacteria. The impact of pufLM gene sequences for studies on the environmental diversity of phototrophic bacteria is discussed and the possibility of their identification on the species level in environmental samples is pointed out. PMID:29472894

Packaging signals in two single-stranded RNA viruses imply a conserved assembly mechanism and geometry of the packaged genome.

PubMed

Dykeman, Eric C; Stockley, Peter G; Twarock, Reidun

2013-09-09

The current paradigm for assembly of single-stranded RNA viruses is based on a mechanism involving non-sequence-specific packaging of genomic RNA driven by electrostatic interactions. Recent experiments, however, provide compelling evidence for sequence specificity in this process both in vitro and in vivo. The existence of multiple RNA packaging signals (PSs) within viral genomes has been proposed, which facilitates assembly by binding coat proteins in such a way that they promote the protein-protein contacts needed to build the capsid. The binding energy from these interactions enables the confinement or compaction of the genomic RNAs. Identifying the nature of such PSs is crucial for a full understanding of assembly, which is an as yet untapped potential drug target for this important class of pathogens. Here, for two related bacterial viruses, we determine the sequences and locations of their PSs using Hamiltonian paths, a concept from graph theory, in combination with bioinformatics and structural studies. Their PSs have a common secondary structure motif but distinct consensus sequences and positions within the respective genomes. Despite these differences, the distributions of PSs in both viruses imply defined conformations for the packaged RNA genomes in contact with the protein shell in the capsid, consistent with a recent asymmetric structure determination of the MS2 virion. The PS distributions identified moreover imply a preferred, evolutionarily conserved assembly pathway with respect to the RNA sequence with potentially profound implications for other single-stranded RNA viruses known to have RNA PSs, including many animal and human pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

CHROMA: consensus-based colouring of multiple alignments for publication.

PubMed

Goodstadt, L; Ponting, C P

2001-09-01

CHROMA annotates multiple protein sequence alignments by consensus to produce formatted and coloured text suitable for incorporation into other documents for publication. The package is designed to be flexible and reliable, and has a simple-to-use graphical user interface running under Microsoft Windows. Both the executables and source code for CHROMA running under Windows and Linux (portable command-line only) are freely available at http://www.lg.ndirect.co.uk/chroma. Software enquiries should be directed to CHROMA@lg.ndirect.co.uk.

From Principal Component to Direct Coupling Analysis of Coevolution in Proteins: Low-Eigenvalue Modes are Needed for Structure Prediction

PubMed Central

Cocco, Simona; Monasson, Remi; Weigt, Martin

2013-01-01

Various approaches have explored the covariation of residues in multiple-sequence alignments of homologous proteins to extract functional and structural information. Among those are principal component analysis (PCA), which identifies the most correlated groups of residues, and direct coupling analysis (DCA), a global inference method based on the maximum entropy principle, which aims at predicting residue-residue contacts. In this paper, inspired by the statistical physics of disordered systems, we introduce the Hopfield-Potts model to naturally interpolate between these two approaches. The Hopfield-Potts model allows us to identify relevant ‘patterns’ of residues from the knowledge of the eigenmodes and eigenvalues of the residue-residue correlation matrix. We show how the computation of such statistical patterns makes it possible to accurately predict residue-residue contacts with a much smaller number of parameters than DCA. This dimensional reduction allows us to avoid overfitting and to extract contact information from multiple-sequence alignments of reduced size. In addition, we show that low-eigenvalue correlation modes, discarded by PCA, are important to recover structural information: the corresponding patterns are highly localized, that is, they are concentrated in few sites, which we find to be in close contact in the three-dimensional protein fold. PMID:23990764

Accurate prediction of protein–protein interactions from sequence alignments using a Bayesian method

PubMed Central

Burger, Lukas; van Nimwegen, Erik

2008-01-01

Accurate and large-scale prediction of protein–protein interactions directly from amino-acid sequences is one of the great challenges in computational biology. Here we present a new Bayesian network method that predicts interaction partners using only multiple alignments of amino-acid sequences of interacting protein domains, without tunable parameters, and without the need for any training examples. We first apply the method to bacterial two-component systems and comprehensively reconstruct two-component signaling networks across all sequenced bacteria. Comparisons of our predictions with known interactions show that our method infers interaction partners genome-wide with high accuracy. To demonstrate the general applicability of our method we show that it also accurately predicts interaction partners in a recent dataset of polyketide synthases. Analysis of the predicted genome-wide two-component signaling networks shows that cognates (interacting kinase/regulator pairs, which lie adjacent on the genome) and orphans (which lie isolated) form two relatively independent components of the signaling network in each genome. In addition, while most genes are predicted to have only a small number of interaction partners, we find that 10% of orphans form a separate class of ‘hub' nodes that distribute and integrate signals to and from up to tens of different interaction partners. PMID:18277381

Crammed signaling motifs in the T-cell receptor.

PubMed

Borroto, Aldo; Abia, David; Alarcón, Balbino

2014-09-01

Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering. Copyright © 2014 Elsevier B.V. All rights reserved.

Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

PubMed

Batchu, Navish Kumar; Khater, Shradha; Patil, Sonal; Nagle, Vinod; Das, Gautam; Bhadra, Bhaskar; Sapre, Ajit; Dasgupta, Santanu

2018-03-05

A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites. Copyright © 2018 Elsevier Inc. All rights reserved.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

PubMed

Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair

2016-01-01

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts

PubMed Central

Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W.; Eyun, Seong-il; Noriega, Daniel D.; Siegfried, Blair

2016-01-01

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest. PMID:26949943

Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales.

PubMed

Prakash, Hariprasath; Rudramurthy, Shivaprakash Mandya; Gandham, Prasad S; Ghosh, Anup Kumar; Kumar, Milner M; Badapanda, Chandan; Chakrabarti, Arunaloke

2017-09-18

Apophysomyces species are prevalent in tropical countries and A. variabilis is the second most frequent agent causing mucormycosis in India. Among Apophysomyces species, A. elegans, A. trapeziformis and A. variabilis are commonly incriminated in human infections. The genome sequences of A. elegans and A. trapeziformis are available in public database, but not A. variabilis. We, therefore, performed the whole genome sequence of A. variabilis to explore its genomic structure and possible genes determining the virulence of the organism. The whole genome of A. variabilis NCCPF 102052 was sequenced and the genomic structure of A. variabilis was compared with already available genome structures of A. elegans, A. trapeziformis and other medically important Mucorales. The total size of genome assembly of A. variabilis was 39.38 Mb with 12,764 protein-coding genes. The transposable elements (TEs) were low in Apophysomyces genome and the retrotransposon Ty3-gypsy was the common TE. Phylogenetically, Apophysomyces species were grouped closely with Phycomyces blakesleeanus. OrthoMCL analysis revealed 3025 orthologues proteins, which were common in those three pathogenic Apophysomyces species. Expansion of multiple gene families/duplication was observed in Apophysomyces genomes. Approximately 6% of Apophysomyces genes were predicted to be associated with virulence on PHIbase analysis. The virulence determinants included the protein families of CotH proteins (invasins), proteases, iron utilisation pathways, siderophores and signal transduction pathways. Serine proteases were the major group of proteases found in all Apophysomyces genomes. The carbohydrate active enzymes (CAZymes) constitute the majority of the secretory proteins. The present study is the maiden attempt to sequence and analyze the genomic structure of A. variabilis. Together with available genome sequence of A. elegans and A. trapeziformis, the study helped to indicate the possible virulence determinants of pathogenic Apophysomyces species. The presence of unique CAZymes in cell wall might be exploited in future for antifungal drug development.

Discovering Motifs in Biological Sequences Using the Micron Automata Processor.

PubMed

Roy, Indranil; Aluru, Srinivas

2016-01-01

Finding approximately conserved sequences, called motifs, across multiple DNA or protein sequences is an important problem in computational biology. In this paper, we consider the (l, d) motif search problem of identifying one or more motifs of length l present in at least q of the n given sequences, with each occurrence differing from the motif in at most d substitutions. The problem is known to be NP-complete, and the largest solved instance reported to date is (26,11). We propose a novel algorithm for the (l,d) motif search problem using streaming execution over a large set of non-deterministic finite automata (NFA). This solution is designed to take advantage of the micron automata processor, a new technology close to deployment that can simultaneously execute multiple NFA in parallel. We demonstrate the capability for solving much larger instances of the (l, d) motif search problem using the resources available within a single automata processor board, by estimating run-times for problem instances (39,18) and (40,17). The paper serves as a useful guide to solving problems using this new accelerator technology.

Using hidden Markov models to align multiple sequences.

PubMed

Mount, David W

2009-07-01

A hidden Markov model (HMM) is a probabilistic model of a multiple sequence alignment (msa) of proteins. In the model, each column of symbols in the alignment is represented by a frequency distribution of the symbols (called a "state"), and insertions and deletions are represented by other states. One moves through the model along a particular path from state to state in a Markov chain (i.e., random choice of next move), trying to match a given sequence. The next matching symbol is chosen from each state, recording its probability (frequency) and also the probability of going to that state from a previous one (the transition probability). State and transition probabilities are multiplied to obtain a probability of the given sequence. The hidden nature of the HMM is due to the lack of information about the value of a specific state, which is instead represented by a probability distribution over all possible values. This article discusses the advantages and disadvantages of HMMs in msa and presents algorithms for calculating an HMM and the conditions for producing the best HMM.

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

PubMed

Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

2015-11-01

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

PubMed

Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

2014-03-28

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

PubMed

Warfield, Linda; Tuttle, Lisa M; Pacheco, Derek; Klevit, Rachel E; Hahn, Steven

2014-08-26

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

Information Propagation in Developmental Enhancers

NASA Astrophysics Data System (ADS)

Jena, Siddhartha; Levine, Michael

Rather than encoding information about protein sequence, certain lengths of noncoding DNA, called enhancers, interact with protein machinery such as transcription factors to precisely regulate gene expression. Enhancers have been studied extensively in the fruit fly Drosophila melanogaster, where they regulate the expression of developmental genes that establish the blueprint of the adult fly. It has been suggested that enhancer sequences possess a specific but unknown syntax with regards to the placement and strength of transcription factor binding sites. Moreover, studies in divergent fly species have shown that compensatory evolution allows for maintenance of enhancer functionality despite considerable variation in primary DNA sequence. Here, the possible role of enhancers as signal processing modules is studied as a way of explaining these two findings. We first demonstrate how this framework can be used to explain the fine-tuned spatiotemporal dynamics of gene expression. We then explore the evolutionary pressure on enhancer sequences and the resulting emergence of enhancers that are linked by compensatory mutations. This study provides a possible mechanism for the function of multiple enhancers linked to a single gene.

[Advance of the study on LRRK2 gene in Parkinson's disease].

PubMed

Zhang, Yu; Chen, Shengdi

2008-12-01

The leucine-rich repeat kinase2 (LRRK2) has been identified to be the gene causing autosomal dominant inherited Parkinson's disease(PD)8. The clinical features of this type of PD are similar to those of idiopathic PD, but the pathological changes are diverse. The mutation types and frequencies of the LRRK2 distribute unevenly in different populations. LRRK2 is a large complex protein with multiple functions and expresses widely in human body. Sequence alignment shows that LRRK2 might be a multiple function kinase for substrate phosphorylation and might also act as a scaffolding protein. Further study on the physiological function and pathogenic mechanism of LRRK2 will help to find out the possible pathogenesis and new treatment for PD.

P³DB 3.0: From plant phosphorylation sites to protein networks.

PubMed

Yao, Qiuming; Ge, Huangyi; Wu, Shangquan; Zhang, Ning; Chen, Wei; Xu, Chunhui; Gao, Jianjiong; Thelen, Jay J; Xu, Dong

2014-01-01

In the past few years, the Plant Protein Phosphorylation Database (P(3)DB, http://p3db.org) has become one of the most significant in vivo data resources for studying plant phosphoproteomics. We have substantially updated P(3)DB with respect to format, new datasets and analytic tools. In the P(3)DB 3.0, there are altogether 47 923 phosphosites in 16 477 phosphoproteins curated across nine plant organisms from 32 studies, which have met our multiple quality standards for acquisition of in vivo phosphorylation site data. Centralized by these phosphorylation data, multiple related data and annotations are provided, including protein-protein interaction (PPI), gene ontology, protein tertiary structures, orthologous sequences, kinase/phosphatase classification and Kinase Client Assay (KiC Assay) data--all of which provides context for the phosphorylation event. In addition, P(3)DB 3.0 incorporates multiple network viewers for the above features, such as PPI network, kinase-substrate network, phosphatase-substrate network, and domain co-occurrence network to help study phosphorylation from a systems point of view. Furthermore, the new P(3)DB reflects a community-based design through which users can share datasets and automate data depository processes for publication purposes. Each of these new features supports the goal of making P(3)DB a comprehensive, systematic and interactive platform for phosphoproteomics research.

Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders.

PubMed

Craig, Catherine L; Riekel, Christian

2002-12-01

The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera larvae) are made up of hierarchically organized, repetitive arrays of amino acids. Fhc fibroin genes are characterized by a similar molecular genetic architecture of two exons and one intron, but the organization and size of these units differs. The Flag, Ser (sericin gene) and BR (Balbiani ring genes; both fibrous proteins) genes are made up of multiple exons and introns. Sequences coding for crystalline and non-crystalline protein domains are integrated in the repetitive regions of Fhc and MA exons, but not in the protein glues Ser1 and BR-1. Genetic 'hot-spots' promote recombination errors in Fhc, MA, and Flag. Codon bias, structural constraint, point mutations, and shortened coding arrays may be alternative means of stabilizing precursor mRNA transcripts. Differential regulation of gene expression and selective splicing of the mRNA transcript may allow rapid adaptation of silk functional properties to different physical environments.

Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design.

PubMed

Thyme, Summer B; Baker, David; Bradley, Philip

2012-06-08

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. Published by Elsevier Ltd.

A novel BLAST-Based Relative Distance (BBRD) method can effectively group members of protein arginine methyltransferases and suggest their evolutionary relationship.

PubMed

Wang, Yi-Chun; Wang, Jing-Doo; Chen, Chin-Han; Chen, Yi-Wen; Li, Chuan

2015-03-01

We developed a novel BLAST-Based Relative Distance (BBRD) method by Pearson's correlation coefficient to avoid the problems of tedious multiple sequence alignment and complicated outgroup selection. We showed its application on reconstructing reliable phylogeny for nucleotide and protein sequences as exemplified by the fmr-1 gene and dihydrolipoamide dehydrogenase, respectively. We then used BBRD to resolve 124 protein arginine methyltransferases (PRMTs) that are homologues of nine mammalian PRMTs. The tree placed the uncharacterized PRMT9 with PRMT7 in the same clade, outside of all the Type I PRMTs including PRMT1 and its vertebrate paralogue PRMT8, PRMT3, PRMT6, PRMT2 and PRMT4. The PRMT7/9 branch then connects with the type II PRMT5. Some non-vertebrates contain different PRMTs without high sequence homology with the mammalian PRMTs. For example, in the case of Drosophila arginine methyltransferase (DART) and Trypanosoma brucei methyltransferases (TbPRMTs) in the analyses, the BBRD program grouped them with specific clades and thus suggested their evolutionary relationships. The BBRD method thus provided a great tool to construct a reliable tree for members of protein families through evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

Rab protein evolution and the history of the eukaryotic endomembrane system

PubMed Central

Brighouse, Andrew; Dacks, Joel B.

2010-01-01

Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity. PMID:20582450

Ectopic Expression of Capsicum-Specific Cell Wall Protein Capsicum annuum Senescence-Delaying 1 (CaSD1) Delays Senescence and Induces Trichome Formation in Nicotiana benthamiana

PubMed Central

Seo, Eunyoung; Yeom, Seon-In; Jo, SungHwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-01-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation. PMID:22441673

MassSieve: Panning MS/MS peptide data for proteins

PubMed Central

Slotta, Douglas J.; McFarland, Melinda A.; Markey, Sanford P.

2010-01-01

We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. PMID:20564260

Identification and characterisation of the angiotensin converting enzyme-3 (ACE3) gene: a novel mammalian homologue of ACE

PubMed Central

Rella, Monika; Elliot, Joann L; Revett, Timothy J; Lanfear, Jerry; Phelan, Anne; Jackson, Richard M; Turner, Anthony J; Hooper, Nigel M

2007-01-01

Background Mammalian angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation. Although multiple ACE-like proteins exist in non-mammalian organisms, to date only one other ACE homologue, ACE2, has been identified in mammals. Results Here we report the identification and characterisation of the gene encoding a third homologue of ACE, termed ACE3, in several mammalian genomes. The ACE3 gene is located on the same chromosome downstream of the ACE gene. Multiple sequence alignment and molecular modelling have been employed to characterise the predicted ACE3 protein. In mouse, rat, cow and dog, the predicted protein has mutations in some of the critical residues involved in catalysis, including the catalytic Glu in the HEXXH zinc binding motif which is Gln, and ESTs or reverse-transcription PCR indicate that the gene is expressed. In humans, the predicted ACE3 protein has an intact HEXXH motif, but there are other deletions and insertions in the gene and no ESTs have been identified. Conclusion In the genomes of several mammalian species there is a gene that encodes a novel, single domain ACE-like protein, ACE3. In mouse, rat, cow and dog ACE3, the catalytic Glu is replaced by Gln in the putative zinc binding motif, indicating that in these species ACE3 would lack catalytic activity as a zinc metalloprotease. In humans, no evidence was found that the ACE3 gene is expressed and the presence of deletions and insertions in the sequence indicate that ACE3 is a pseudogene. PMID:17597519

The sequence, structure and evolutionary features of HOTAIR in mammals

PubMed Central

2011-01-01

Background An increasing number of long noncoding RNAs (lncRNAs) have been identified recently. Different from all the others that function in cis to regulate local gene expression, the newly identified HOTAIR is located between HoxC11 and HoxC12 in the human genome and regulates HoxD expression in multiple tissues. Like the well-characterised lncRNA Xist, HOTAIR binds to polycomb proteins to methylate histones at multiple HoxD loci, but unlike Xist, many details of its structure and function, as well as the trans regulation, remain unclear. Moreover, HOTAIR is involved in the aberrant regulation of gene expression in cancer. Results To identify conserved domains in HOTAIR and study the phylogenetic distribution of this lncRNA, we searched the genomes of 10 mammalian and 3 non-mammalian vertebrates for matches to its 6 exons and the two conserved domains within the 1800 bp exon6 using Infernal. There was just one high-scoring hit for each mammal, but many low-scoring hits were found in both mammals and non-mammalian vertebrates. These hits and their flanking genes in four placental mammals and platypus were examined to determine whether HOTAIR contained elements shared by other lncRNAs. Several of the hits were within unknown transcripts or ncRNAs, many were within introns of, or antisense to, protein-coding genes, and conservation of the flanking genes was observed only between human and chimpanzee. Phylogenetic analysis revealed discrete evolutionary dynamics for orthologous sequences of HOTAIR exons. Exon1 at the 5' end and a domain in exon6 near the 3' end, which contain domains that bind to multiple proteins, have evolved faster in primates than in other mammals. Structures were predicted for exon1, two domains of exon6 and the full HOTAIR sequence. The sequence and structure of two fragments, in exon1 and the domain B of exon6 respectively, were identified to robustly occur in predicted structures of exon1, domain B of exon6 and the full HOTAIR in mammals. Conclusions HOTAIR exists in mammals, has poorly conserved sequences and considerably conserved structures, and has evolved faster than nearby HoxC genes. Exons of HOTAIR show distinct evolutionary features, and a 239 bp domain in the 1804 bp exon6 is especially conserved. These features, together with the absence of some exons and sequences in mouse, rat and kangaroo, suggest ab initio generation of HOTAIR in marsupials. Structure prediction identifies two fragments in the 5' end exon1 and the 3' end domain B of exon6, with sequence and structure invariably occurring in various predicted structures of exon1, the domain B of exon6 and the full HOTAIR. PMID:21496275

Ankyrin-repeat containing proteins of microbes: a conserved structure with functional diversity

PubMed Central

Al-Khodor, Souhaila; Price, Christopher T.; Kalia, Awdhesh; Kwaik, Yousef Abu

2009-01-01

Summary The ankyrin repeat (ANK) is the most common protein-protein interaction motif in nature and predominantly found in eukaryotic proteins. The genome sequencing of various pathogenic or symbiotic bacteria and eukaryotic viruses identified numerous genes encoding ANK-containing proteins that were proposed to have been acquired from eukaryotes by horizontal gene transfer. However, the recent discovery of additional ANK-containing proteins encoded in the genomes of archaea and free-living bacteria suggests either a more ancient origin of the ANK motif or multiple convergent evolution events. Many bacterial pathogens employ various types of secretion systems to deliver ANK-containing proteins into eukaryotic cells where they mimic or manipulate various host functions. Understanding the molecular and biochemical functions of this family of proteins will enhance our understanding of important host-microbe interactions. PMID:19962898

Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics.

PubMed

Deutsch, Eric W; Sun, Zhi; Campbell, David S; Binz, Pierre-Alain; Farrah, Terry; Shteynberg, David; Mendoza, Luis; Omenn, Gilbert S; Moritz, Robert L

2016-11-04

The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances-a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ∼20,000 primary isoforms plus contaminants to a very large database that includes almost all nonredundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the discovered peptides against a more complex database. We have set up an automated system that downloads all the source databases on the first of each month and automatically generates a new set of search databases and makes them available for download at http://www.peptideatlas.org/thisp/ .

Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics

PubMed Central

Deutsch, Eric W.; Sun, Zhi; Campbell, David S.; Binz, Pierre-Alain; Farrah, Terry; Shteynberg, David; Mendoza, Luis; Omenn, Gilbert S.; Moritz, Robert L.

2016-01-01

The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances – a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ~20,000 primary isoforms plus contaminants to a very large database that includes almost all non-redundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the discovered peptides against a more complex database. We have set up an automated system that downloads all the source databases on the first of each month and automatically generates a new set of search databases and makes them available for download at http://www.peptideatlas.org/thisp/. PMID:27577934

First principles design of a core bioenergetic transmembrane electron-transfer protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteinsmore » and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.« less

A Dual Interaction Between the 5'- and 3'-Ends of the Melon Necrotic Spot Virus (MNSV) RNA Genome Is Required for Efficient Cap-Independent Translation.

PubMed

Miras, Manuel; Rodríguez-Hernández, Ana M; Romero-López, Cristina; Berzal-Herranz, Alfredo; Colchero, Jaime; Aranda, Miguel A; Truniger, Verónica

2018-01-01

In eukaryotes, the formation of a 5'-cap and 3'-poly(A) dependent protein-protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3'-CITE (for cap-independent translation enhancer), a RNA element present in their 3'-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3'-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5'- and 3'-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3'-CITE to the 5'-end. For the carmovirus melon necrotic spot virus (MNSV), a 3'-CITE has been identified, and the presence of its 5'-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5'-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3'-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3'-CITE and at least one complementary sequence in the 5'-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5'-3' RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.

The CanOE strategy: integrating genomic and metabolic contexts across multiple prokaryote genomes to find candidate genes for orphan enzymes.

PubMed

Smith, Adam Alexander Thil; Belda, Eugeni; Viari, Alain; Medigue, Claudine; Vallenet, David

2012-05-01

Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes), a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short). The first step locates "genomic metabolons", i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12.

Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

PubMed

Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

1997-06-15

Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

PubMed

Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

PubMed Central

Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

2014-01-01

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

Prediction of protein-protein interactions from amino acid sequences with ensemble extreme learning machines and principal component analysis.

PubMed

You, Zhu-Hong; Lei, Ying-Ke; Zhu, Lin; Xia, Junfeng; Wang, Bing

2013-01-01

Protein-protein interactions (PPIs) play crucial roles in the execution of various cellular processes and form the basis of biological mechanisms. Although large amount of PPIs data for different species has been generated by high-throughput experimental techniques, current PPI pairs obtained with experimental methods cover only a fraction of the complete PPI networks, and further, the experimental methods for identifying PPIs are both time-consuming and expensive. Hence, it is urgent and challenging to develop automated computational methods to efficiently and accurately predict PPIs. We present here a novel hierarchical PCA-EELM (principal component analysis-ensemble extreme learning machine) model to predict protein-protein interactions only using the information of protein sequences. In the proposed method, 11188 protein pairs retrieved from the DIP database were encoded into feature vectors by using four kinds of protein sequences information. Focusing on dimension reduction, an effective feature extraction method PCA was then employed to construct the most discriminative new feature set. Finally, multiple extreme learning machines were trained and then aggregated into a consensus classifier by majority voting. The ensembling of extreme learning machine removes the dependence of results on initial random weights and improves the prediction performance. When performed on the PPI data of Saccharomyces cerevisiae, the proposed method achieved 87.00% prediction accuracy with 86.15% sensitivity at the precision of 87.59%. Extensive experiments are performed to compare our method with state-of-the-art techniques Support Vector Machine (SVM). Experimental results demonstrate that proposed PCA-EELM outperforms the SVM method by 5-fold cross-validation. Besides, PCA-EELM performs faster than PCA-SVM based method. Consequently, the proposed approach can be considered as a new promising and powerful tools for predicting PPI with excellent performance and less time.

Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses.

PubMed

Sheth, Bhavisha P; Thaker, Vrinda S

2015-10-01

Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools. The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder. The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica. A strategy as used here, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel rapid and economic procedure, especially in cases when protein coding loci are considered. Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. A herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. An integrated approach using DNA barcoding and structural analyses was carried out to identify the herbal powder. The herbal powder was identified as Cassia javanica L.

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yeming; Opperman, Laura; Wickens, Marvin

2011-11-02

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short regionmore » of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.« less

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yeming; Opperman, Laura; Wickens, Marvin

2010-08-19

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short regionmore » of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.« less

The SpTransformer Gene Family (Formerly Sp185/333) in the Purple Sea Urchin and the Functional Diversity of the Anti-Pathogen rSpTransformer-E1 Protein

PubMed Central

Smith, L. Courtney; Lun, Cheng Man

2017-01-01

The complex innate immune system of sea urchins is underpinned by several multigene families including the SpTransformer family (SpTrf; formerly Sp185/333) with estimates of ~50 members, although the family size is likely variable among individuals of Strongylocentrotus purpuratus. The genes are small with similar structure, are tightly clustered, and have several types of repeats in the second of two exons and that surround each gene. The density of repeats suggests that the genes are positioned within regions of genomic instability, which may be required to drive sequence diversification. The second exon encodes the mature protein and is composed of blocks of sequence called elements that are present in mosaics of defined element patterns and are the major source of sequence diversity. The SpTrf genes respond swiftly to immune challenge, but only a single gene is expressed per phagocyte. Many of the mRNAs appear to be edited and encode proteins with altered and/or missense sequence that are often truncated, of which some may be functional. The standard SpTrf protein structure is an N-terminal glycine-rich region, a central RGD motif, a histidine-rich region, and a C-terminal region. Function is predicted from a recombinant protein, rSpTransformer-E1 (rSpTrf-E1), which binds to Vibrio and Saccharomyces, but not to Bacillus, and binds tightly to lipopolysaccharide, β-1,3-glucan, and flagellin, but not to peptidoglycan. rSpTrf-E1 is intrinsically disordered but transforms to α helical structure in the presence of binding targets including lipopolysaccharide, which may underpin the characteristics of binding to multiple targets. SpTrf proteins associate with coelomocyte membranes, and rSpTrf-E1 binds specifically to phosphatidic acid (PA). When rSpTrf-E1 is bound to PA in liposome membranes, it induces morphological changes in liposomes that correlate with PA clustering and leakage of luminal contents, and it extracts or removes PA from the bilayer. The multitasking activities of rSpTrf-E1 infer multiple and perhaps overlapping activities for the hundreds of native SpTrf proteins that are produced by individual sea urchins. This likely generates a flexible and highly protective immune system for the sea urchin in its marine habitat that it shares with broad arrays of microbes that may be pathogens and opportunists. PMID:28713368

Probing sequence dependence of folding pathway of α-helix bundle proteins through free energy landscape analysis.

PubMed

Shao, Qiang

2014-06-05

A comparative study on the folding of multiple three-α-helix bundle proteins including α3D, α3W, and the B domain of protein A (BdpA) is presented. The use of integrated-tempering-sampling molecular dynamics simulations achieves reversible folding and unfolding events in individual short trajectories, which thus provides an efficient approach to sufficiently sample the configuration space of protein and delineate the folding pathway of α-helix bundle. The detailed free energy landscape analyses indicate that the folding mechanism of α-helix bundle is not uniform but sequence dependent. A simple model is then proposed to predict folding mechanism of α-helix bundle on the basis of amino acid composition: α-helical proteins containing higher percentage of hydrophobic residues than charged ones fold via nucleation-condensation mechanism (e.g., α3D and BdpA) whereas proteins having opposite tendency in amino acid composition more likely fold via the framework mechanism (e.g., α3W). The model is tested on various α-helix bundle proteins, and the predicted mechanism is similar to the most approved one for each protein. In addition, the common features in the folding pathway of α-helix bundle protein are also deduced. In summary, the present study provides comprehensive, atomic-level picture of the folding of α-helix bundle proteins.

Predicting permanent and transient protein-protein interfaces.

PubMed

La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

2013-05-01

Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. Copyright © 2013 Wiley Periodicals, Inc.

MitoNuc: a database of nuclear genes coding for mitochondrial proteins. Update 2002.

PubMed

Attimonelli, Marcella; Catalano, Domenico; Gissi, Carmela; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Santamaria, Monica; Pesole, Graziano; Saccone, Cecilia

2002-01-01

Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organelle genomes. Mitochondrial genomes have been extensively sequenced and analysed and data have been collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc, a database containing detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa. The MitoNuc database can be retrieved through SRS and is available via the web site http://bighost.area.ba.cnr.it/mitochondriome where other mitochondrial databases developed by our group, the complete list of the sequenced mitochondrial genomes, links to other mitochondrial sites and related information, are available. The MitoAln database, related to MitoNuc in the previous release, reporting the multiple alignments of the relevant homologous protein coding regions, is no longer supported in the present release. In order to keep the links among entries in MitoNuc from homologous proteins, a new field in the database has been defined: the cluster identifier, an alpha numeric code used to identify each cluster of homologous proteins. A comment field derived from the corresponding SWISS-PROT entry has been introduced; this reports clinical data related to dysfunction of the protein. The logic scheme of MitoNuc database has been implemented in the ORACLE DBMS. This will allow the end-users to retrieve data through a friendly interface that will be soon implemented.

Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

PubMed Central

Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh M. B.; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B.; Claesson, Marcus J.; Ross, R. Paul; Scott, Karen P.; O'Toole, Paul W.

2013-01-01

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes. PMID:23935906

The actin multigene family and livestock speciation using the polymerase chain reaction.

PubMed

Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

1998-01-01

Actins constitute a family of highly-conserved multifunctional intracellular proteins, best known as myofibrillar components in striated muscle fibres. Most vertebrate genomes contain numerous actin genes with high sequence homology in protein coding regions but considerable variability in intron number and sizes. This genetic diversity can be utilised for livestock speciation purposes. The high sequence conservation has enabled a single pair of oligonucleotides to be used to prime the polymerase chain reaction (PCR) with DNA extracted from all animals so far studied. Multiple amplification products were obtained which on gel electrophoresis constituted characteristic species-specific 'fingerprints'. The patterns were reproducible, did not vary between individuals of the same breed or between different breeds within a species, and could be generated even from heat-processed muscle held at 120 degrees C for one hour. Given the capacity of PCR to amplify relatively short sequences in highly-degraded DNA, this approach may be suitable for authentication of processed meat products.

Protein alignment algorithms with an efficient backtracking routine on multiple GPUs.

PubMed

Blazewicz, Jacek; Frohmberg, Wojciech; Kierzynka, Michal; Pesch, Erwin; Wojciechowski, Pawel

2011-05-20

Pairwise sequence alignment methods are widely used in biological research. The increasing number of sequences is perceived as one of the upcoming challenges for sequence alignment methods in the nearest future. To overcome this challenge several GPU (Graphics Processing Unit) computing approaches have been proposed lately. These solutions show a great potential of a GPU platform but in most cases address the problem of sequence database scanning and computing only the alignment score whereas the alignment itself is omitted. Thus, the need arose to implement the global and semiglobal Needleman-Wunsch, and Smith-Waterman algorithms with a backtracking procedure which is needed to construct the alignment. In this paper we present the solution that performs the alignment of every given sequence pair, which is a required step for progressive multiple sequence alignment methods, as well as for DNA recognition at the DNA assembly stage. Performed tests show that the implementation, with performance up to 6.3 GCUPS on a single GPU for affine gap penalties, is very efficient in comparison to other CPU and GPU-based solutions. Moreover, multiple GPUs support with load balancing makes the application very scalable. The article shows that the backtracking procedure of the sequence alignment algorithms may be designed to fit in with the GPU architecture. Therefore, our algorithm, apart from scores, is able to compute pairwise alignments. This opens a wide range of new possibilities, allowing other methods from the area of molecular biology to take advantage of the new computational architecture. Performed tests show that the efficiency of the implementation is excellent. Moreover, the speed of our GPU-based algorithms can be almost linearly increased when using more than one graphics card.

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

USDA-ARS?s Scientific Manuscript database

Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel transcript which is represented by multiple ESTs derived only from the oocyte c...

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics.

PubMed

Niu, Xiaogang; Ding, Jienv; Zhang, Wenbo; Li, Qianwen; Hu, Yunfei; Jin, Changwen

2018-06-01

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15 N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15 N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1 H- 15 N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR.

PubMed

Lee, Soon Goo; Krishnan, Hari B; Jez, Joseph M

2014-04-29

The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.

Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

PubMed

Abraham, Anup; Chandler, Douglas E

2017-10-01

Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

PubMed

Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

2016-05-21

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

ExDom: an integrated database for comparative analysis of the exon–intron structures of protein domains in eukaryotes

PubMed Central

Bhasi, Ashwini; Philip, Philge; Manikandan, Vinu; Senapathy, Periannan

2009-01-01

We have developed ExDom, a unique database for the comparative analysis of the exon–intron structures of 96 680 protein domains from seven eukaryotic organisms (Homo sapiens, Mus musculus, Bos taurus, Rattus norvegicus, Danio rerio, Gallus gallus and Arabidopsis thaliana). ExDom provides integrated access to exon-domain data through a sophisticated web interface which has the following analytical capabilities: (i) intergenomic and intragenomic comparative analysis of exon–intron structure of domains; (ii) color-coded graphical display of the domain architecture of proteins correlated with their corresponding exon-intron structures; (iii) graphical analysis of multiple sequence alignments of amino acid and coding nucleotide sequences of homologous protein domains from seven organisms; (iv) comparative graphical display of exon distributions within the tertiary structures of protein domains; and (v) visualization of exon–intron structures of alternative transcripts of a gene correlated to variations in the domain architecture of corresponding protein isoforms. These novel analytical features are highly suited for detailed investigations on the exon–intron structure of domains and make ExDom a powerful tool for exploring several key questions concerning the function, origin and evolution of genes and proteins. ExDom database is freely accessible at: http://66.170.16.154/ExDom/. PMID:18984624

Amino acid sequence analysis of the annexin super-gene family of proteins.

PubMed

Barton, G J; Newman, R H; Freemont, P S; Crumpton, M J

1991-06-15

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.

The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

PubMed

Potapova, Ulyana; Feranchuk, Sergey; Leonova, Galina; Belikov, Sergei

2018-03-01

In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

PubMed

Vasquez, Kevin A; Hatridge, Taylor A; Curtis, Nicholas C; Contreras, Lydia M

2016-02-19

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Benchmark analysis of native and artificial NAD+-dependent enzymes generated by a sequence based design method with or without phylogenetic data.

PubMed

Nakano, Shogo; Motoyama, Tomoharu; Miyashita, Yurina; Ishizuka, Yuki; Matsuo, Naoya; Tokiwa, Hiroaki; Shinoda, Suguru; Asano, Yasuhisa; Ito, Sohei

2018-05-22

The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial L-threonine 3-dehydrogenase (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1 and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH): the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5°C higher than CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were two- and seven-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

The GAGA protein of Drosophila is phosphorylated by CK2.

PubMed

Bonet, Carles; Fernández, Irene; Aran, Xavier; Bernués, Jordi; Giralt, Ernest; Azorín, Fernando

2005-08-19

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.

Optimal de novo design of MRM experiments for rapid assay development in targeted proteomics.

PubMed

Bertsch, Andreas; Jung, Stephan; Zerck, Alexandra; Pfeifer, Nico; Nahnsen, Sven; Henneges, Carsten; Nordheim, Alfred; Kohlbacher, Oliver

2010-05-07

Targeted proteomic approaches such as multiple reaction monitoring (MRM) overcome problems associated with classical shotgun mass spectrometry experiments. Developing MRM quantitation assays can be time consuming, because relevant peptide representatives of the proteins must be found and their retention time and the product ions must be determined. Given the transitions, hundreds to thousands of them can be scheduled into one experiment run. However, it is difficult to select which of the transitions should be included into a measurement. We present a novel algorithm that allows the construction of MRM assays from the sequence of the targeted proteins alone. This enables the rapid development of targeted MRM experiments without large libraries of transitions or peptide spectra. The approach relies on combinatorial optimization in combination with machine learning techniques to predict proteotypicity, retention time, and fragmentation of peptides. The resulting potential transitions are scheduled optimally by solving an integer linear program. We demonstrate that fully automated construction of MRM experiments from protein sequences alone is possible and over 80% coverage of the targeted proteins can be achieved without further optimization of the assay.

Transmembrane peptides as sensors of the membrane physical state

NASA Astrophysics Data System (ADS)

Piotto, Stefano; Di Biasi, Luigi; Sessa, Lucia; Concilio, Simona

2018-05-01

Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state suggested a new approach for the discovery of antimicrobial peptides.

Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science.

PubMed

Giessen, Tobias W

2016-10-01

Compartmentalization is one of the defining features of life. Cells use protein compartments to exert spatial control over their metabolism, store nutrients and create unique microenvironments needed for essential physiological processes. Encapsulins are a recently discovered class of protein nanocompartments found in bacteria and archaea that naturally encapsulate cargo proteins. A short C-terminal targeting sequence directs the highly specific encapsulation process in vivo. Here, I will initially discuss the properties, diversity and putative function of encapsulins. The unique characteristics and potential uses of the self-sorting cargo-packaging process found in encapsulin systems will then be highlighted. Examples for the application of encapsulins as cell-specific optical nanoprobes and targeted therapeutic delivery systems will be discussed with an emphasis on the ability to integrate multiple functionalities within a single nanodevice. By fusing targeting sequences to non-native proteins, encapsulins can also be used as specific nanocontainers and enzymatic nanoreactors in vivo. I will end by briefly discussing future avenues for encapsulin research related to both basic microbial metabolism and applications in biomedicine, catalysis and materials science. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene.

PubMed

Bogado, A L G; Martins, G F; Sasse, J P; Guimarães, J da S; Garcia, J L

2017-03-15

Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.

MALDI Top-Down sequencing: calling N- and C-terminal protein sequences with high confidence and speed.

PubMed

Suckau, Detlev; Resemann, Anja

2009-12-01

The ability to match Top-Down protein sequencing (TDS) results by MALDI-TOF to protein sequences by classical protein database searching was evaluated in this work. Resulting from these analyses were the protein identity, the simultaneous assignment of the N- and C-termini and protein sequences of up to 70 residues from either terminus. In combination with de novo sequencing using the MALDI-TDS data, even fusion proteins were assigned and the detailed sequence around the fusion site was elucidated. MALDI-TDS allowed to efficiently match protein sequences quickly and to validate recombinant protein structures-in particular, protein termini-on the level of undigested proteins.

A novel sodium bicarbonate cotransporter-like gene in an ancient duplicated region: SLC4A9 at 5q31

PubMed Central

Lipovich, Leonard; Lynch, Eric D; Lee, Ming K; King, Mary-Claire

2001-01-01

Background: Sodium bicarbonate cotransporter (NBC) genes encode proteins that execute coupled Na+ and HCO3- transport across epithelial cell membranes. We report the discovery, characterization, and genomic context of a novel human NBC-like gene, SLC4A9, on chromosome 5q31. Results: SLC4A9 was initially discovered by genomic sequence annotation and further characterized by sequencing of long-insert cDNA library clones. The predicted protein of 990 amino acids has 12 transmembrane domains and high sequence similarity to other NBCs. The 23-exon gene has 14 known mRNA isoforms. In three regions, mRNA sequence variation is generated by the inclusion or exclusion of portions of an exon. Noncoding SLC4A9 cDNAs were recovered multiple times from different libraries. The 3' untranslated region is fragmented into six alternatively spliced exons and contains expressed Alu, LINE and MER repeats. SLC4A9 has two alternative stop codons and six polyadenylation sites. Its expression is largely restricted to the kidney. In silico approaches were used to characterize two additional novel SLC4A genes and to place SLC4A9 within the context of multiple paralogous gene clusters containing members of the epidermal growth factor (EGF), ankyrin (ANK) and fibroblast growth factor (FGF) families. Seven human EGF-SLC4A-ANK-FGF clusters were found. Conclusion: The novel sodium bicarbonate cotransporter-like gene SLC4A9 demonstrates abundant alternative mRNA processing. It belongs to a growing class of functionally diverse genes characterized by inefficient highly variable splicing. The evolutionary history of the EGF-SLC4A-ANK-FGF gene clusters involves multiple rounds of duplication, apparently followed by large insertions and deletions at paralogous loci and genome-wide gene shuffling. PMID:11305939

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2.

PubMed

Bubenik, Jodi L; Miniard, Angela C; Driscoll, Donna M

2014-01-01

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3/Moat-D) Is Expressed in the SAE Squalus acanthias Shark Embryo–Derived Cell Line

PubMed Central

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2008-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E. PMID:18284333

Multidrug resistance-associated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark embryo-derived cell line.

PubMed

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2007-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E.

Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

PubMed Central

RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

2007-01-01

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

ExoLocator--an online view into genetic makeup of vertebrate proteins.

PubMed

Khoo, Aik Aun; Ogrizek-Tomas, Mario; Bulovic, Ana; Korpar, Matija; Gürler, Ece; Slijepcevic, Ivan; Šikic, Mile; Mihalek, Ivana

2014-01-01

ExoLocator (http://exolocator.eopsf.org) collects in a single place information needed for comparative analysis of protein-coding exons from vertebrate species. The main source of data--the genomic sequences, and the existing exon and homology annotation--is the ENSEMBL database of completed vertebrate genomes. To these, ExoLocator adds the search for ostensibly missing exons in orthologous protein pairs across species, using an extensive computational pipeline to narrow down the search region for the candidate exons and find a suitable template in the other species, as well as state-of-the-art implementations of pairwise alignment algorithms. The resulting complements of exons are organized in a way currently unique to ExoLocator: multiple sequence alignments, both on the nucleotide and on the peptide levels, clearly indicating the exon boundaries. The alignments can be inspected in the web-embedded viewer, downloaded or used on the spot to produce an estimate of conservation within orthologous sets, or functional divergence across paralogues.