Mohtar, M Aiman; Hernychova, Lenka; O'Neill, J Robert; Lawrence, Melanie L; Murray, Euan; Vojtesek, Borek; Hupp, Ted R
2018-04-01
AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.
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.
L1-CAM and N-CAM: From Adhesion Proteins to Pharmacological Targets.
Colombo, Federico; Meldolesi, Jacopo
2015-11-01
L1 cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (N-CAM), key members of the immunoglobulin-like CAM (Ig-CAM) family, were first recognized to play critical roles in surface interactions of neurons, by binding with each other and with extracellular matrix (ECM) proteins. Subsequently, adhesion was recognized to include signaling due to both activation of β-integrin, with the generation of intracellular cascades, and integration with the surface cytoskeleton. The importance of the two Ig-CAMs was revealed by their activation of the tyrosine kinase receptors of fibroblast growth factor (FGF), epidermal growth factor (EGF), and nerve growth factor (NGF). Based on these complex signaling properties, L1-CAM and N-CAM have become of great potential pharmacological interest in neurons and cancers. Treatment of neurodegenerative disorders and cognitive deficits of neurons is aimed to increase the cell Ig-CAM tone, possibly provided by synthetic/mimetic peptides. In cancer cells, where Ig-CAMs are often overexpressed, the proteins are employed for prognosis. The approaches to therapy are based on protein downregulation, antibodies, and adoptive immunotherapy. Copyright © 2015 Elsevier Ltd. All rights reserved.
NASA Astrophysics Data System (ADS)
Ezerski, Jacob; Cheung, Margaret
CaM forms distinct conformation states through modifications in its charge distribution upon binding to Ca2+ ions. The occurrence of protein structural change resulting from an altered charge distribution is paramount in the scheme of cellular signaling. Not only is charge induced structural change observed in CaM, it is also seen in an essential binding target: calmodulin-depended protein kinase II (CaMKII). In order to investigate the mechanism of selectivity in relation to changes in secondary structure, the CaM binding domain of CaMKII is isolated. Experimentally, charged residues of the CaMKII peptide are systematically mutated to alanine, resulting in altered binding kinetics between the peptide and the Ca2+ saturated state of CaM. We perform an all atom simulation of the wildtype (RRK) and mutated (AAA) CaMKII peptides and generate structures from the trajectory. We analyze RRK and AAA using DSSP and find significant structural differences due to the mutation. Structures from the RRK and AAA ensembles are then selected and docked onto the crystal structure of Ca2+ saturated CaM. We observe that RRK binds to CaM at the C-terminus, whereas the 3-residue mutation, AAA, shows increased patterns of binding to the N-terminus and linker regions of CaM. Due to the conformational change of the peptide ensemble from charged residue mutation, a distinct change in the binding site can be seen, which offers an explanation to experimentally observed changes in kinetic binding rates
NASA Technical Reports Server (NTRS)
Reddy, A. S.; Reddy, V. S.; Golovkin, M.
2000-01-01
Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.
Isolation and characterization of a novel calmodulin-binding protein from potato
NASA Technical Reports Server (NTRS)
Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.
2002-01-01
Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.
A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats
NASA Technical Reports Server (NTRS)
Safadi, F.; Reddy, V. S.; Reddy, A. S.
2000-01-01
Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.
Howard, John; Finch, Nicole A; Ochrietor, Judith D
2010-07-01
The purpose of this study was to determine the binding affinities of Basigin gene products and neural cell adhesion molecule L1cam for monocarboxylate transporter-1 (MCT1). ELISA binding assays were performed in which recombinant proteins of the transmembrane domains of Basigin gene products and L1cam were incubated with MCT1 captured from mouse brain. It was determined that Basigin gene products bind MCT1 with moderate affinity, but L1cam does not bind MCT1. Despite a high degree of sequence conservation between Basigin gene products and L1cam, the sequences are different enough to prevent L1cam from interacting with MCT1.
Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.
Westerlund, Annie M; Delemotte, Lucie
2018-04-01
Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.
Effect of Ca2+ on the promiscuous target-protein binding of calmodulin
Westerlund, Annie M.
2018-01-01
Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity. PMID:29614072
Photounbinding of Calmodulin from a Family of CaM Binding Peptides
Neumüller, Klaus G.; Elsayad, Kareem; Reisecker, Johannes M.; Waxham, M. Neal; Heinze, Katrin G.
2010-01-01
Background Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. Methodology/Principal Findings We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner. PMID:21124984
Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L
NASA Technical Reports Server (NTRS)
Ling, V.; Assmann, S. M.
1992-01-01
The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.
Wu, Dan; Peng, Xuan; Liu, Xu; Zhang, Jiaojiao; Zhao, Junfeng; Chen, Kunming; Zhao, Liqun
2016-01-01
Salt is a major threat to plant growth and crop productivity. Calmodulin (CaM), the most important multifunctional Ca2+ sensor protein in plants, mediates reactions against environmental stresses through target proteins; however, direct proof of the participation of CaM in salt tolerance and its corresponding signaling pathway in vivo is lacking. In this study, we found that AtCaM1 and AtCaM4 produced salt-responsive CaM isoforms according to real-time reverse transcription-polymerase chain reaction analyses; this result was verified based on a phenotypic analysis of salt-treated loss-of-function mutant and transgenic plants. We also found that the level of nitric oxide (NO), an important salt-responsive signaling molecule, varied in response to salt treatment depending on AtCaM1 and AtCaM4 expression. GSNOR is considered as an important and widely utilized regulatory component of NO homeostasis in plant resistance protein signaling networks. In vivo and in vitro protein-protein interaction assays revealed direct binding between AtCaM4 and S-nitrosoglutathione reductase (GSNOR), leading to reduced GSNOR activity and an increased NO level. Overexpression of GSNOR intensified the salt sensitivity of cam4 mutant plants accompanied by a reduced internal NO level, whereas a gsnor deficiency increased the salt tolerance of cam4 plants accompanied by an increased internal NO level. Physiological experiments showed that CaM4-GSNOR, acting through NO, reestablished the ion balance to increase plant resistance to salt stress. Together, these data suggest that AtCaM1 and AtCaM4 serve as signals in plant salt resistance by promoting NO accumulation through the binding and inhibition of GSNOR. This could be a conserved defensive signaling pathway in plants and animals. PMID:27684709
Davis, J Q; Bennett, V
1994-11-04
Neurofascin, L1, NrCAM, NgCAM, and neuroglian are membrane-spanning cell adhesion molecules with conserved cytoplasmic domains that are believed to play important roles in development of the nervous system. This report presents biochemical evidence that the cytoplasmic domains of these molecules associate directly with ankyrins, a family of spectrin-binding proteins located on the cytoplasmic surface of specialized plasma membrane domains. Rat neurofascin and NrCAM together comprise over 0.5% of the membrane protein in adult brain tissue. Linkage of these ankyrin-binding cell adhesion molecules to spectrin-based structures may provide a major class of membrane-cytoskeletal connections in adult brain as well as earlier stages of development.
Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein*
Vlach, Jiri; Samal, Alexandra B.; Saad, Jamil S.
2014-01-01
Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8–43 (MA-(8–43)). Here, we present the NMR structure of CaM bound to MA-(8–43). Our data revealed that MA-(8–43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8–43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8–43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication. PMID:24500712
Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.
2007-01-01
Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592
Genes encoding calmodulin-binding proteins in the Arabidopsis genome
NASA Technical Reports Server (NTRS)
Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.
2002-01-01
Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.
Nucleic acid encoding DS-CAM proteins and products related thereto
Korenberg, Julie R.
2005-11-01
In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.
Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein
NASA Technical Reports Server (NTRS)
Yang, T.; Poovaiah, B. W.
2000-01-01
Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.
MIPS: a calmodulin-binding protein of Gracilaria lemaneiformis under heat shock.
Zhang, Xuan; Zhou, Huiyue; Zang, Xiaonan; Gong, Le; Sun, Hengyi; Zhang, Xuecheng
2014-08-01
To study the Ca(2+)/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5'RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61-67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.
Ochiai, Nagahiro; Masumoto, Shuji; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Yamauchi, Takashi
2007-05-01
We previously found the neuronal cell-type specific promoter and binding partner of the beta isoform of Ca(2+)/calmodulin-dependent protein kinase II (beta CaM kinase II) in rat brain [Donai, H., Morinaga, H., Yamauchi, T., 2001. Genomic organization and neuronal cell type specific promoter activity of beta isoform of Ca(2+)/calmodulin-dependent protein kinase II of rat brain. Mol. Brain Res. 94, 35-47]. In the present study, we purified a protein that binds specifically a promoter region of beta CaM kinase II gene from a nuclear extract of the rat cerebellum using DEAE-cellulose column chromatography, ammonium sulfate fractionation, gel filtration and polyacrylamide gel electrophoresis. The purified protein was identified as rat leucine-rich protein 157 (rLRP157) using tandem mass spectrometry. Then, we prepared its cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly(A)(+)RNA of rat cerebellum. The rLRP157 cDNA was introduced into mouse neuroblastomaxrat glioma hybrid NG108-15 cells, and cells stably expressing rLRP157 (NG/LRP cells) were isolated. Binding of rLRP157 with the promoter sequence was confirmed by electrophoretic mobility shift assay using nuclear extract of NG/LRP cells. A luciferase reporter gene containing a promoter of beta CaM kinase II was transiently expressed in NG/LRP cells. Under the conditions, the promoter activity was enhanced about 2.6-fold in NG/LRP cells as compared with wild-type cells. The expression of rLRP157 mRNA was paralleled with that of beta CaM kinase II in the adult and embryo rat brain detected by in situ hybridization. Nuclear localization of rLRP157 was confirmed using GFP-rLRP157 fusion protein investigated under a confocal microscope. These results indicate that rLRP157 is one of the proteins binding to, and regulating the activity of, the promoter of beta CaM kinase II.
THE INTERACTION BETWEEN L1-TYPE PROTEINS AND ANKYRINS - A MASTER SWITCH FOR L1-TYPE CAM FUNCTION #
HORTSCH, MICHAEL; NAGARAJ, KAKANAHALLI; GODENSCHWEGE, TANJA A.
2008-01-01
L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that Ankyrin-binding to L1-type CAMs provides a master switch. The interaction with Ankyrins directs L1-type adhesive proteins into different functional contexts, either Ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into Ankyrin-dependent functions, such as L1’s role at axon initial segments (AIS), paranodal regions, synapses and in dendrites. PMID:18839070
NASA Technical Reports Server (NTRS)
Baird, R. A.; Steyger, P. S.; Schuff, N. R.
1997-01-01
Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.
Udani, M; Zen, Q; Cottman, M; Leonard, N; Jefferson, S; Daymont, C; Truskey, G; Telen, M J
1998-01-01
Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors. PMID:9616226
BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.
Fischer, Cornelia; Sauter, Margret; Dietrich, Petra
2017-01-01
Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.
Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
Abraham, Paul E.; Yin, Hengfu; Borland, Anne M.; ...
2016-11-21
Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of photosynthesis that maximizes water-use efficiency (WUE) via an inverse (compared to C 3 and C 4 photosynthesis-performing species) day/night pattern of stomatal closure/opening to shift CO 2 uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behavior that underpins CAM. Here, we report high-resolution temporal behaviors of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare those observations to the well-established C 3 modelmore » plant, Arabidopsis thaliana. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis thaliana. Moreover, we identified widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet how this molecular timekeeping regulates CAM physiology remains unclear. In this paper, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets together provide a resource to inform efforts to engineer more water-use efficient CAM pathway traits into economically valuable C 3 crops.« less
Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
DOE Office of Scientific and Technical Information (OSTI.GOV)
Abraham, Paul E.; Yin, Hengfu; Borland, Anne M.
Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of photosynthesis that maximizes water-use efficiency (WUE) via an inverse (compared to C 3 and C 4 photosynthesis-performing species) day/night pattern of stomatal closure/opening to shift CO 2 uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behavior that underpins CAM. Here, we report high-resolution temporal behaviors of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare those observations to the well-established C 3 modelmore » plant, Arabidopsis thaliana. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis thaliana. Moreover, we identified widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet how this molecular timekeeping regulates CAM physiology remains unclear. In this paper, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets together provide a resource to inform efforts to engineer more water-use efficient CAM pathway traits into economically valuable C 3 crops.« less
Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I
2014-01-01
Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426
Cooperative Allosteric Ligand Binding in Calmodulin
NASA Astrophysics Data System (ADS)
Nandigrami, Prithviraj
Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds
Characterization of Novel Calmodulin Binding Domains within IQ Motifs of IQGAP1
Jang, Deok-Jin; Ban, Byungkwan; Lee, Jin-A
2011-01-01
IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known calmodulin (CaM) binding protein, is involved in a wide range of cellular processes including cell proliferation, tumorigenesis, adhesion, and migration. Interaction of IQGAP1 with CaM is important for its cellular functions. Although each IQ domain of IQGAP1 for CaM binding has been characterized in a Ca2+-dependent or -independent manner, it was not clear which IQ motifs are physiologically relevant for CaM binding in the cells. In this study, we performed immunoprecipitation using 3xFLAGhCaM in mammalian cell lines to characterize the domains of IQGAP1 that are key for CaM binding under physiological conditions. Interestingly, using this method, we identified two novel domains, IQ(2.7-3) and IQ(3.5-4.4), within IQGAP1 that were involved in Ca2+-independent or -dependent CaM binding, respectively. Mutant analysis clearly showed that the hydrophobic regions within IQ(2.7-3) were mainly involved in apoCaM binding, while the basic amino acids and hydrophobic region of IQ(3.5-4.4) were required for Ca2+/CaM binding. Finally, we showed that IQ(2.7-3) was the main apoCaM binding domain and both IQ(2.7-3) and IQ(3.5-4.4) were required for Ca2+/CaM binding within IQ(1- 2-3-4). Thus, we identified and characterized novel direct CaM binding motifs essential for IQGAP1. This finding indicates that IQGAP1 plays a dynamic role via direct interactions with CaM in a Ca2+-dependent or -independent manner. PMID:22080369
Patel, Neal M.; Kinzer-Ursem, Tamara L.
2017-01-01
A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca2+) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca2+/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca2+ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca2+/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca2+/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca2+, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca2+ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the in vivo frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca2+/Ca
Liu, Yanshun; Zheng, Xunhai; Mueller, Geoffrey A.; Sobhany, Mack; DeRose, Eugene F.; Zhang, Yingpei; London, Robert E.; Birnbaumer, Lutz
2012-01-01
Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp76 of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1. PMID:23109337
Neurogranin alters the structure and calcium binding properties of calmodulin.
Hoffman, Laurel; Chandrasekar, Anuja; Wang, Xu; Putkey, John A; Waxham, M Neal
2014-05-23
Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process
Miao, Yinglong; Baudry, Jerome
2011-01-01
Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431
Fogel, Adam I; Li, Yue; Giza, Joanna; Wang, Qing; Lam, Tukiet T; Modis, Yorgo; Biederer, Thomas
2010-11-05
Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.
Fogel, Adam I.; Li, Yue; Giza, Joanna; Wang, Qing; Lam, TuKiet T.; Modis, Yorgo; Biederer, Thomas
2010-01-01
Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn60. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn70/Asn104 flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn60 reduces adhesion, N-glycans at Asn70/Asn104 of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion. PMID:20739279
González-Andrade, Martin; Rodríguez-Sotres, Rogelio; Madariaga-Mazón, Abraham; Rivera-Chávez, José; Mata, Rachel; Sosa-Peinado, Alejandro; Del Pozo-Yauner, Luis; Arias-Olguín, Imilla I
2016-01-01
In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca(2+)-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the "open" and "closed" conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM's inhibitors correlated well with available experimental data as the r(2) obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca(2+)-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca(2+)-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca(2+)-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.
Ababou, Abdessamad; Zaleska, Mariola
2015-12-01
Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins. Copyright © 2015 Elsevier Inc. All rights reserved.
Identification of the Calmodulin-Binding Domains of Fas Death Receptor
Chang, Bliss J.; Samal, Alexandra B.; Vlach, Jiri; Fernandez, Timothy F.; Brooke, Dewey; Prevelige, Peter E.; Saad, Jamil S.
2016-01-01
The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas–mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209–239 (Fas-Pep1) and 251–288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD–CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling
Calmodulin and calmodulin-binding proteins in cystic fibrosis and normal human fibroblasts
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tallant, E.A.; Wallace, R.W.
1986-05-01
The authors have investigated the possibility that a lesion in a calmodulin (CaM)-dependent regulatory mechanism may be involved in cystic fibrosis (CF). The level of CaM, CaM-binding proteins (CaM-BP) and a CaM-dependent phosphatase (CaM-Ptase) have been compared in cultured fibroblasts from CF patients versus age- and sex-matched control subjects. The CaM concentration, measured by radioimmunoassay, ranged from 0.20 to 0.76 ..mu..g/mg protein (n=8); there was no significant difference in the average CaM concentration from CF patients vs controls. Using Western blotting techniques with /sup 125/I-CaM, they detected at least ten distinct CaM-BPs in fibroblasts with molecular weights ranging from 230Kmore » to 37K; the only consistent difference between control and CF cell lines was in a 46.5K CaM-BP, which was depressed in all three CF samples. The 46.5 K CaM-BP was found only in the particulate fraction. A 59K CaM-BP was identified as a CaM-Ptase by its crossreactivity with an antibody against a brain CaM-Ptase. There was no significant difference in CaM-Ptase activity or in the amount of the phosphatase as determined by radioimmunoassay in CF vs. normal samples (n=8). Thus, the level of CaM as well as its various enzymes and proteins do not appear to be altered in CF fibroblasts except for a CaM-BP of 46.5K, the identity of which is currently being investigated.« less
Schanzer, Juergen M; Fichtner, Iduna; Baeuerle, Patrick A; Kufer, Peter
2006-01-01
Cytokine targeting to tumor-associated antigens via antibody cytokine fusion proteins has demonstrated potent antitumor activity in numerous animal models and has led to the clinical development of 2 antibody-interleukin-2 (IL-2) fusion proteins. We previously reported on the construction and in vitro properties of a "dual" cytokine fusion protein for simultaneous targeted delivery of human granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-2 to human tumors. The fusion protein is based on a heterodimerized core structure formed by human CH1 and Ckappa domains (heterominibody) with C-terminally fused human cytokines and N-terminally fused single-chain antibody fragments specific for the tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM). For testing the antitumor activity in syngeneic mouse xenograft models, we developed "dual cytokine heterominibodies" with murine cytokines (mDCH). mDCH fusion proteins and, as controls, "single cytokine heterominibodies" (SCH) carrying either murine GM-CSF (mGM-CSF) or murine IL-2 (mIL-2) were constructed, of which all retained the specific activities of cytokines and binding to the Ep-CAM antigen on human Ep-CAM transfected mouse colon carcinoma CT26-KSA cells. Over a 5-day treatment course, DCH fusion proteins induced significant inhibition of established pulmonary CT26-KSA metastases in immune-competent Balb/c mice at low daily doses of 1 mug of fusion protein per mouse. However, with the tested dosing schemes, antitumor activity of mDCH was largely independent of cytokine targeting to tumors as demonstrated by a control protein with mutated Ep-CAM binding sites. Single cytokine fusion proteins mSCH-GM-CSF and mSCH-IL-2 showed similar antitumor activity as the dual cytokine fusion protein mDCH, indicating that GM-CSF and IL-2 in one molecule did not significantly synergize in tumor rejection under our experimental conditions. Our results seem to contradict the notion that IL-2 and GM
Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations.
Kukic, Predrag; Kannan, Arvind; Dijkstra, Maurits J J; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele
2015-10-01
It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a 'tube model' approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the 'CamTube' force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost.
Mapping the Protein Fold Universe Using the CamTube Force Field in Molecular Dynamics Simulations
Dijkstra, Maurits J. J.; Abeln, Sanne; Camilloni, Carlo; Vendruscolo, Michele
2015-01-01
It has been recently shown that the coarse-graining of the structures of polypeptide chains as self-avoiding tubes can provide an effective representation of the conformational space of proteins. In order to fully exploit the opportunities offered by such a ‘tube model’ approach, we present here a strategy to combine it with molecular dynamics simulations. This strategy is based on the incorporation of the ‘CamTube’ force field into the Gromacs molecular dynamics package. By considering the case of a 60-residue polyvaline chain, we show that CamTube molecular dynamics simulations can comprehensively explore the conformational space of proteins. We obtain this result by a 20 μs metadynamics simulation of the polyvaline chain that recapitulates the currently known protein fold universe. We further show that, if residue-specific interaction potentials are added to the CamTube force field, it is possible to fold a protein into a topology close to that of its native state. These results illustrate how the CamTube force field can be used to explore efficiently the universe of protein folds with good accuracy and very limited computational cost. PMID:26505754
Interaction entropy for protein-protein binding
NASA Astrophysics Data System (ADS)
Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.
2017-03-01
Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.
Interaction entropy for protein-protein binding.
Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H
2017-03-28
Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.
Conformational heterogeneity of the calmodulin binding interface
NASA Astrophysics Data System (ADS)
Shukla, Diwakar; Peck, Ariana; Pande, Vijay S.
2016-04-01
Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.
Regulation of Polycystin-1 Function by Calmodulin Binding
Doerr, Nicholas; Wang, Yidi; Kipp, Kevin R.; Liu, Guangyi; Benza, Jesse J.; Pletnev, Vladimir; Pavlov, Tengis S.; Staruschenko, Alexander; Mohieldin, Ashraf M.; Takahashi, Maki; Nauli, Surya M.; Weimbs, Thomas
2016-01-01
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM. PMID:27560828
Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A
2013-01-01
A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.
Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A.
2013-01-01
A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis. PMID:24155914
Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations.
Rydzewski, J; Nowak, W
2017-08-10
Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B' helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.
Calmodulin is a phospholipase C-beta interacting protein.
McCullar, Jennifer S; Larsen, Shana A; Millimaki, Ryan A; Filtz, Theresa M
2003-09-05
Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.
Mosolits, Szilvia; Markovic, Katja; Fagerberg, Jan; Frödin, Jan-Erik; Rezvany, Mohammad-Reza; Kiaii, Shahryar; Mellstedt, Håkan; Jeddi-Tehrani, Mahmood
2005-06-01
The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-gamma production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-gamma T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.
Sankpal, NV; Willman, MW; Fleming, TP; Mayfield, J; Gillanders, WE
2014-01-01
p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. PMID:19141643
Kim, Jiyoung; Sung, Gi-Ho
2018-03-19
Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca 2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca 2+ signals are partially decoded by Ca 2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca 2+ -dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca 2+ . In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421-2538) binds to CaM in a Ca 2+ -dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.
Wei, Qing; La, David; Kihara, Daisuke
2017-01-01
Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .
De Fusco, Claudia; Brear, Paul; Iegre, Jessica; Georgiou, Kathy Hadje; Sore, Hannah F; Hyvönen, Marko; Spring, David R
2017-07-01
Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors. Copyright © 2017. Published by Elsevier Ltd.
Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chen, Baowei; Lowry, David; Mayer, M. Uljana
2008-08-09
The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H- 15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH.more » Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ± 0.04 μM), which results in a reduction in the rate of ReAsH binding from 4900 M -1 sec -1 to 370 M -1 sec -1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ± 3 μM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain
Churn, S B; DeLorenzo, R J
1998-10-26
gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.
Ca2+/calmodulin binding to PSD-95 mediates homeostatic synaptic scaling down.
Chowdhury, Dhrubajyoti; Turner, Matthew; Patriarchi, Tommaso; Hergarden, Anne C; Anderson, David; Zhang, Yonghong; Sun, Junqing; Chen, Chao-Yin; Ames, James B; Hell, Johannes W
2018-01-04
Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca 2+ influx, Ca 2+ /calmodulin (Ca 2+ /CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca 2+ /CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM R 126E , as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca 2+ /CaM to PSD-95 induced by a chronic increase in Ca 2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission. © 2017 The Authors.
Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian
2014-01-01
The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790
Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8
2012-01-01
Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca2+ ions. These two lobes have differing affinities for Ca2+, and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca2+ binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles. PMID:22971080
Detecting cis-regulatory binding sites for cooperatively binding proteins
van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves
2008-01-01
Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778
Oshiro, Satoshi; Honda, Shinya
2014-04-18
Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.
Stauch, Kelly; Kieken, Fabien; Sorgen, Paul
2012-01-01
In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca2+, pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis. PMID:22718765
NASA Technical Reports Server (NTRS)
Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia
2002-01-01
Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.
Abiotic stress responses in plants: roles of calmodulin-regulated proteins.
Virdi, Amardeep S; Singh, Supreet; Singh, Prabhjeet
2015-01-01
Intracellular changes in calcium ions (Ca(2+)) in response to different biotic and abiotic stimuli are detected by various sensor proteins in the plant cell. Calmodulin (CaM) is one of the most extensively studied Ca(2+)-sensing proteins and has been shown to be involved in transduction of Ca(2+) signals. After interacting with Ca(2+), CaM undergoes conformational change and influences the activities of a diverse range of CaM-binding proteins. A number of CaM-binding proteins have also been implicated in stress responses in plants, highlighting the central role played by CaM in adaptation to adverse environmental conditions. Stress adaptation in plants is a highly complex and multigenic response. Identification and characterization of CaM-modulated proteins in relation to different abiotic stresses could, therefore, prove to be essential for a deeper understanding of the molecular mechanisms involved in abiotic stress tolerance in plants. Various studies have revealed involvement of CaM in regulation of metal ions uptake, generation of reactive oxygen species and modulation of transcription factors such as CAMTA3, GTL1, and WRKY39. Activities of several kinases and phosphatases have also been shown to be modulated by CaM, thus providing further versatility to stress-associated signal transduction pathways. The results obtained from contemporary studies are consistent with the proposed role of CaM as an integrator of different stress signaling pathways, which allows plants to maintain homeostasis between different cellular processes. In this review, we have attempted to present the current state of understanding of the role of CaM in modulating different stress-regulated proteins and its implications in augmenting abiotic stress tolerance in plants.
Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald
2014-12-10
A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.
Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua
2014-10-01
Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.
In Situ Protein Binding Assay Using Fc-Fusion Proteins.
Padmanabhan, Nirmala; Siddiqui, Tabrez J
2017-01-01
This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.
Davis, J Q; McLaughlin, T; Bennett, V
1993-04-01
A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and
Predicting protein-binding RNA nucleotides with consideration of binding partners.
Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook
2015-06-01
In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in
Functions of Intracellular Retinoid Binding-Proteins.
Napoli, Joseph L
Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.
Abiotic stress responses in plants: roles of calmodulin-regulated proteins
Virdi, Amardeep S.; Singh, Supreet; Singh, Prabhjeet
2015-01-01
Intracellular changes in calcium ions (Ca2+) in response to different biotic and abiotic stimuli are detected by various sensor proteins in the plant cell. Calmodulin (CaM) is one of the most extensively studied Ca2+-sensing proteins and has been shown to be involved in transduction of Ca2+ signals. After interacting with Ca2+, CaM undergoes conformational change and influences the activities of a diverse range of CaM-binding proteins. A number of CaM-binding proteins have also been implicated in stress responses in plants, highlighting the central role played by CaM in adaptation to adverse environmental conditions. Stress adaptation in plants is a highly complex and multigenic response. Identification and characterization of CaM-modulated proteins in relation to different abiotic stresses could, therefore, prove to be essential for a deeper understanding of the molecular mechanisms involved in abiotic stress tolerance in plants. Various studies have revealed involvement of CaM in regulation of metal ions uptake, generation of reactive oxygen species and modulation of transcription factors such as CAMTA3, GTL1, and WRKY39. Activities of several kinases and phosphatases have also been shown to be modulated by CaM, thus providing further versatility to stress-associated signal transduction pathways. The results obtained from contemporary studies are consistent with the proposed role of CaM as an integrator of different stress signaling pathways, which allows plants to maintain homeostasis between different cellular processes. In this review, we have attempted to present the current state of understanding of the role of CaM in modulating different stress-regulated proteins and its implications in augmenting abiotic stress tolerance in plants. PMID:26528296
Fogel, Adam I; Stagi, Massimiliano; Perez de Arce, Karen; Biederer, Thomas
2011-09-16
Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.
Calcium-binding proteins and development
NASA Technical Reports Server (NTRS)
Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)
1998-01-01
The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.
Engineered proteins as specific binding reagents.
Binz, H Kaspar; Plückthun, Andreas
2005-08-01
Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.
Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya
2014-07-01
Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less
Kitani, Takako; Okuno, Sachiko; Takeuchi, Masayuki; Fujisawa, Hitoshi
2003-07-01
Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.
Erythropoietin binding protein from mammalian serum
Clemons, Gisela K.
1997-01-01
Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.
González-Andrade, Martín; Figueroa, Mario; Rodríguez-Sotres, Rogelio; Mata, Rachel; Sosa-Peinado, Alejandro
2009-04-01
This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ( approximately 80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC(50) values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.
SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.
Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W
2016-10-20
RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.
Erythropoietin binding protein from mammalian serum
Clemons, G.K.
1997-04-29
Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.
[Glutamate-binding membrane proteins from human platelets].
Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A
1991-09-01
Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.
Protein Binding: Do We Ever Learn?▿
Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula
2011-01-01
Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013
Identification of AOSC-binding proteins in neurons
NASA Astrophysics Data System (ADS)
Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu
2008-11-01
Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.
The pineapple genome and the evolution of CAM photosynthesis
Ming, Ray; VanBuren, Robert; Wai, Ching Man; Tang, Haibao; Schatz, Michael C.; Bowers, John E.; Lyons, Eric; Wang, Ming-Li; Chen, Jung; Biggers, Eric; Zhang, Jisen; Huang, Lixian; Zhang, Lingmao; Miao, Wenjing; Zhang, Jian; Ye, Zhangyao; Miao, Chenyong; Lin, Zhicong; Wang, Hao; Zhou, Hongye; Yim, Won C.; Priest, Henry D.; Zheng, Chunfang; Woodhouse, Margaret; Edger, Patrick P.; Guyot, Romain; Guo, Hao-Bo; Guo, Hong; Zheng, Guangyong; Singh, Ratnesh; Sharma, Anupma; Min, Xiangjia; Zheng, Yun; Lee, Hayan; Gurtowski, James; Sedlazeck, Fritz J.; Harkess, Alex; McKain, Michael R.; Liao, Zhenyang; Fang, Jingping; Liu, Juan; Zhang, Xiaodan; Zhang, Qing; Hu, Weichang; Qin, Yuan; Wang, Kai; Chen, Li-Yu; Shirley, Neil; Lin, Yann-Rong; Liu, Li-Yu; Hernandez, Alvaro G.; Wright, Chris L.; Bulone, Vincent; Tuskan, Gerald A.; Heath, Katy; Zee, Francis; Moore, Paul H.; Sunkar, Ramanjulu; Leebens-Mack, James H.; Mockler, Todd; Bennetzen, Jeffrey L.; Freeling, Michael; Sankoff, David; Paterson, Andrew H.; Zhu, Xinguang; Yang, Xiaohan; Smith, J. Andrew C.; Cushman, John C.; Paull, Robert E.; Yu, Qingyi
2016-01-01
Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water use efficiency, and the second most important tropical fruit after banana in terms of international trade. We sequenced the genomes of pineapple varieties ‘F153’ and ‘MD2’, and a wild pineapple relative A. bracteatus accession CB5. The pineapple genome has one fewer ancient whole genome duplications than sequenced grass genomes and, therefore, provides an important reference for elucidating gene content and structure in the last common ancestor of extant members of the grass family (Poaceae). Pineapple has a conserved karyotype with seven pre rho duplication chromosomes that are ancestral to extant grass karyotypes. The pineapple lineage has transitioned from C3 photosynthesis to CAM with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues using beta-carbonic anhydrase (βCA) for initial capture of CO2. Promoter regions of all three βCA genes contain a CCA1 binding site that can bind circadian core oscillators. CAM pathway genes were enriched with cis-regulatory elements including the morning (CCACAC) and evening (AAAATATC) elements associated with regulation of circadian-clock genes, providing the first link between CAM and the circadian clock regulation. Gene-interaction network analysis revealed both activation and repression of regulatory elements that control key enzymes in CAM photosynthesis, indicating that CAM evolved by reconfiguration of pathways preexisting in C3 plants. Pineapple CAM photosynthesis is the result of regulatory neofunctionalization of preexisting gene copies and not acquisition of neofunctionalized genes via whole genome or tandem gene duplication. PMID:26523774
Monoclonal antibodies to human vitamin D-binding protein.
Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F
1985-01-01
Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035
Modeling Ionization Events iduced by Protein Protein Binding
NASA Astrophysics Data System (ADS)
Mitra, Rooplekha; Shyam, Radhey; Alexov, Emil
2009-11-01
The association of two or more biological macromolecules dramatically change the environment of the amino acids situated at binding interface and could change ionization states of titratable groups. The change of ionization due to the binding results in proton uptake/release and causes pH-dependence of the binding free energy. We apply computational method, as implemented in Multi Conformation Continuum Electrostatics (MCCE) algorithm, to study protonation evens on a large set of protein-protein complexes. Our results indicate that proton uptake/release is a common phenomena in protein binding since in vast majority of the cases (70%) the binding caused at least 0.5 units proton change. The proton uptake/release was further investigated with respect to interfacial area and charges of the monomers and it was found that macroscopic characteristics are not important determinants. Instead, charge complementarity across the interface and the number of unpaired ionizable groups at the interface are the primary source of proton uptake/release.
Odorant-binding proteins from a primitive termite.
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.
Computational search for aflatoxin binding proteins
NASA Astrophysics Data System (ADS)
Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.
2017-10-01
Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.
NASA Astrophysics Data System (ADS)
Hoegg-Beiler, Maja B.; Sirisi, Sònia; Orozco, Ian J.; Ferrer, Isidre; Hohensee, Svea; Auberson, Muriel; Gödde, Kathrin; Vilches, Clara; de Heredia, Miguel López; Nunes, Virginia; Estévez, Raúl; Jentsch, Thomas J.
2014-03-01
Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2’s biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.
Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima
2017-04-03
The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.
2003-01-01
Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is
Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.
Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver
2018-06-15
It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.
Bryan, R T; Regan, H L; Pirrie, S J; Devall, A J; Cheng, K K; Zeegers, M P; James, N D; Knowles, M A; Ward, D G
2015-03-17
Better biomarkers must be found to develop clinically useful urine tests for bladder cancer. Proteomics can be used to identify the proteins released by cancer cell lines and generate candidate markers for developing such tests. We used shotgun proteomics to identify proteins released into culture media by eight bladder cancer cell lines. These data were compared with protein expression data from the Human Protein Atlas. Epidermal growth factor receptor (EGFR) was identified as a candidate biomarker and measured by ELISA in urine from 60 noncancer control subjects and from 436 patients with bladder cancer and long-term clinical follow-up. Bladder cancer cell lines shed soluble EGFR ectodomain. Soluble EGFR is also detectable in urine and is highly elevated in some patients with high-grade bladder cancer. Urinary EGFR is an independent indicator of poor bladder cancer-specific survival with a hazard ratio of 2.89 (95% CI 1.81-4.62, P<0.001). In multivariable models including both urinary EGFR and EpCAM, both biomarkers are predictive of bladder cancer-specific survival and have prognostic value over and above that provided by standard clinical observations. Measuring urinary EGFR and EpCAM may represent a simple and useful approach for fast-tracking the investigation and treatment of patients with the most aggressive bladder cancers.
Trifluoperazine Regulation of Calmodulin Binding to Fas: A Computational Study
Pan, Di; Yan, Qi; Chen, Yabing; McDonald, Jay M; Song, Yuhua
2011-01-01
Death-inducing signaling complex (DISC) formation is a critical step in Fas-mediated signaling for apoptosis. Previous experiments have demonstrated that the calmodulin (CaM) antagonist, trifluoperazine (TFP) regulates CaM-Fas binding and affects Fas-mediated DISC formation. In this study, we investigated the anti-cooperative characteristics of TFP binding to CaM and the effect of TFP on the CaM-Fas interaction from both structural and thermodynamic perspectives using combined molecular dynamics simulations and binding free energy analyses. We studied the interactions of different numbers of TFP molecules with CaM and explored the effects of the resulting conformational changes in CaM on CaM-Fas binding. Results from these analyses showed that the number of TFP molecules bound to CaM directly influenced α-helix formation and hydrogen bond occupancy within the α-helices of CaM, contributing to the conformational and motion changes in CaM. These changes affected CaM binding to Fas, resulting in secondary structural changes in Fas and conformational and motion changes of Fas in CaM-Fas complexes, potentially perturbing the recruitment of Fas-associated death domain (FADD) for DISC formation. The computational results from this study reveal the structural and molecular mechanisms that underlie the role of the CaM antagonist, TFP, in regulation of CaM-Fas binding and Fas-mediated DISC formation in a concentration-dependent manner. PMID:21656570
Hentz, N G; Daunert, S
1996-11-15
An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.
Clinical role of protein binding of quinolones.
Bergogne-Bérézin, Eugénie
2002-01-01
Protein binding of antibacterials in plasma and tissues has long been considered a component of their pharmacokinetic parameters, playing a potential role in distribution, excretion and therapeutic effectiveness. Since the beginning of the 'antibacterial era', this factor has been extensively analysed for all antibacterial classes, showing that wide variations of the degree of protein binding occur even in the same antibacterial class, as with beta-lactams. As the understanding of protein binding grew, the complexity of the binding system was increasingly perceived and its dynamic character described. Studies of protein binding of the fluoroquinolones have shown that the great majority of these drugs exhibit low protein binding, ranging from approximately 20 to 40% in plasma, and that they are bound predominantly to albumin. The potential role in pharmacokinetics-pharmacodynamics of binding of fluoroquinolones to plasma, tissue and intracellular proteins has been analysed, but it has not been established that protein binding has any significant direct or indirect impact on therapeutic effectiveness. Regarding the factors influencing the tissue distribution of antibacterials, physicochemical characteristics and the small molecular size of fluoroquinolones permit a rapid penetration into extravascular sites and intracellularly, with a rapid equilibrium being established between intravascular and extravascular compartments. The high concentrations of these drugs achieved in tissues, body fluids and intracellularly, in addition to their wide antibacterial spectrum, mean that fluoroquinolones have therapeutic effectiveness in a large variety of infections. The tolerability of quinolones has generally been reported as good, based upon long experience in using pefloxacin, ciprofloxacin and ofloxacin in clinical practice. Among more recently developed molecules, good tolerability has been reported for levofloxacin, moxifloxacin and gatifloxacin, but certain other new
Xu, Xiaojuan; Yano, Masafumi; Uchinoumi, Hitoshi; Hino, Akihiro; Suetomi, Takeshi; Ono, Makoto; Tateishi, Hiroki; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Yamamoto, Takeshi; Ikeda, Yasuhiro; Ikemoto, Noriaki; Matsuzaki, Masunori
2010-01-01
Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca2+ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca2+ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca2+ leak, and then lethal arrhythmia. PMID:20226167
Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.
Kaufmann, Christine; Sauter, Margret
2017-01-01
Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .
VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A
2002-01-01
Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity
The complexity of minocycline serum protein binding.
Zhou, Jian; Tran, Brian T; Tam, Vincent H
2017-06-01
Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum.
Vera-Estrella, Rosario; Barkla, Bronwyn J; Amezcua-Romero, Julio C; Pantoja, Omar
2012-03-01
Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle. © 2011 Blackwell Publishing Ltd.
LiCata, V J; Bernlohr, D A
1998-12-01
Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the
Sterol Carrier Protein-2: Binding Protein for Endocannabinoids
Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.
2015-01-01
The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313
Structure and Function of Lipopolysaccharide Binding Protein
NASA Astrophysics Data System (ADS)
Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.
1990-09-01
The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.
Roles of Copper-Binding Proteins in Breast Cancer.
Blockhuys, Stéphanie; Wittung-Stafshede, Pernilla
2017-04-20
Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.
Subrahmanyam, S; Cronan, J E
1999-01-21
We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.
New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein
Hoffmann, Andreas; Kovermann, Michael; Lilie, Hauke; Fiedler, Markus; Balbach, Jochen; Rudolph, Rainer; Pfeifer, Sven
2012-01-01
A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. PMID:22363609
Factor VII and protein C are phosphatidic acid-binding proteins.
Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H
2013-08-20
Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.
Metal-binding proteins as metal pollution indicators
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hennig, H.F.
1986-03-01
The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effectsmore » on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.« less
Phage display of engineered binding proteins.
Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John
2014-01-01
In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.
Antifreeze Protein Binds Irreversibly to Ice
NASA Astrophysics Data System (ADS)
Braslavsky, I.; Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.
2006-03-01
Many organisms are protected from freezing by antifreeze proteins (AFPs), which bind to ice and prevent its growth by a mechanism not completely understood. Although it has been postulated that AFPs would have to bind irreversibly to arrest the growth of an ice crystal bathed in excess liquid water, the binding forces seem insufficient to support such a tight interaction. By putting a fluorescent tag on a fish AFP, we were able to visualize AFP binding to ice and demonstrate, by lack of recovery after photo-bleaching, that it is indeed irreversible. Because even the most avid protein/ligand interactions exhibit reversibility, this finding is key to understanding the mechanism of antifreeze proteins, which are becoming increasingly valuable in cryopreservation and improving the frost tolerance of crops.
A conserved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.
Godenschwege, Tanja A; Kristiansen, Lars V; Uthaman, Smitha B; Hortsch, Michael; Murphey, Rodney K
2006-01-10
Drosophila Neuroglian (Nrg) and its vertebrate homolog L1-CAM are cell-adhesion molecules (CAM) that have been well studied in early developmental processes. Mutations in the human gene result in a broad spectrum of phenotypes (the CRASH-syndrome) that include devastating neurological disorders such as spasticity and mental retardation. Although the role of L1-CAMs in neurite extension and axon pathfinding has been extensively studied, much less is known about their role in synapse formation. We found that a single extracellular missense mutation in nrg(849) mutants disrupted the physiological function of a central synapse in Drosophila. The identified giant neuron in nrg(849) mutants made a synaptic terminal on the appropriate target, but ultrastructural analysis revealed in the synaptic terminal a dramatic microtubule reduction, which was likely to be the cause for disrupted active zones. Our results reveal that tyrosine phosphorylation of the intracellular ankyrin binding motif was reduced in mutants, and cell-autonomous rescue experiments demonstrated the indispensability of this tyrosine in giant-synapse formation. We also show that this function in giant-synapse formation was conserved in human L1-CAM but neither in human L1-CAM with a pathological missense mutation nor in two isoforms of the paralogs NrCAM and Neurofascin. We conclude that Nrg has a function in synapse formation by organizing microtubules in the synaptic terminal. This novel synaptic function is conserved in human L1-CAM but is not common to all L1-type proteins. Finally, our findings suggest that some aspects of L1-CAM-related neurological disorders in humans may result from a disruption in synapse formation rather than in axon pathfinding.
Theoretical studies of protein-protein and protein-DNA binding rates
NASA Astrophysics Data System (ADS)
Alsallaq, Ramzi A.
Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends
Lactoferrin-binding proteins in Shigella flexneri.
Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S
1992-01-01
The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403
Mapping of ligand-binding cavities in proteins.
Andersson, C David; Chen, Brian Y; Linusson, Anna
2010-05-01
The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterize and map the ligand-binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity, and charge). This approach can provide valuable information on the similarities and dissimilarities, of binding cavities due to mutations, between-species differences and flexibility upon ligand-binding. The presented results show that information on ligand-binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand-binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterization and mapping of "orphan structures", selection of protein structures for docking studies in structure-based design, and identification of proteins for selectivity screens in drug design programs. 2009 Wiley-Liss, Inc.
Mapping of Ligand-Binding Cavities in Proteins
Andersson, C. David; Chen, Brian Y.; Linusson, Anna
2010-01-01
The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterise and map the ligand-binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity and charge). This approach can provide valuable information on the similarities, and dissimilarities, of binding cavities due to mutations, between-species differences and flexibility upon ligand-binding. The presented results show that information on ligand-binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand-binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterisation and mapping of “orphan structures”, selection of protein structures for docking studies in structure-based design and identification of proteins for selectivity screens in drug design programs. PMID:20034113
Sequence-Based Prediction of RNA-Binding Residues in Proteins.
Walia, Rasna R; El-Manzalawy, Yasser; Honavar, Vasant G; Dobbs, Drena
2017-01-01
Identifying individual residues in the interfaces of protein-RNA complexes is important for understanding the molecular determinants of protein-RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein-RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein-RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner.
Tran, Quang-Kim; Vermeer, Mark
2014-01-01
The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific ligands or a Ca(2+)-elevating agent. To confirm direct interaction and locate the calmodulin-binding domain(s), we designed a series of FRET biosensors that consist of enhanced cyan and yellow fluorescent proteins flanking each of GPER's submembrane domains (SMDs). Responses of these biosensors showed that all four submembrane domains directly bind calmodulin. Modifications of biosensor linker identified domains that display the strongest calmodulin-binding affinities and largest biosensor dynamics, including a.a. 83-93, 150-175, 242-259, 330-351, corresponding respectively to SMDs 1, 2, 3, and the juxta-membranous section of SMD4. These biosensors bind calmodulin in a strictly Ca(2+)-dependent fashion and with disparate affinities in the order SMD2>SMD4>SMD3>SMD1, apparent K d values being 0.44 ± 0.03, 1.40 ± 0.16, 8.01 ± 0.29, and 136.62 ± 6.56 µM, respectively. Interestingly, simultaneous determinations of biosensor responses and suitable Ca(2+) indicators identified separate Ca(2+) sensitivities for their interactions with calmodulin. SMD1-CaM complexes display a biphasic Ca(2+) response, representing two distinct species (SMD1 sp1 and SMD1 sp2) with drastically different Ca(2+) sensitivities. The Ca(2+) sensitivities of CaM-SMDs interactions follow the order SMD1sp1>SMD4>SMD2>SMD1sp2>SMD3, EC50(Ca(2+)) values being 0.13 ± 0.02, 0.75 ± 0.05, 2.38 ± 0.13, 3.71 ± 0.13, and 5.15 ± 0.25 µM, respectively. These data indicate that calmodulin may regulate GPER
RNA binding protein and binding site useful for expression of recombinant molecules
Mayfield, Stephen P.
2006-10-17
The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.
RNA binding protein and binding site useful for expression of recombinant molecules
Mayfield, Stephen
2000-01-01
The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.
Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins
NASA Technical Reports Server (NTRS)
Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.
2003-01-01
Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.
Interplay between binding affinity and kinetics in protein-protein interactions.
Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong
2016-07-01
To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus.
Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji
2012-01-01
Thioredoxin binding protein -2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein -2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein -2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein -2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein -2 in metabolic control. Enhancement of thioredoxin binding protein -2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein -2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein -2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β(2)-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.
Sperry, Justin B.; Huang, Richard Y-C.; Zhu, Mei M.; Rempel, Don L.; Gross, Michael L.
2010-01-01
Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the binding affinity of Ca2+ to calmodulin within previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. PMID:21765646
21 CFR 866.5765 - Retinol-binding protein immunological test system.
Code of Federal Regulations, 2011 CFR
2011-04-01
... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...
21 CFR 866.5765 - Retinol-binding protein immunological test system.
Code of Federal Regulations, 2012 CFR
2012-04-01
... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...
21 CFR 866.5765 - Retinol-binding protein immunological test system.
Code of Federal Regulations, 2014 CFR
2014-04-01
... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...
21 CFR 866.5765 - Retinol-binding protein immunological test system.
Code of Federal Regulations, 2013 CFR
2013-04-01
... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...
Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus
Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji
2012-01-01
Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lane, R.P.; Vielmetter, J.; Dreyer, W.J.
1996-08-01
The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less
The MTA family proteins as novel histone H3 binding proteins.
Wu, Meng; Wang, Lina; Li, Qian; Li, Jiwen; Qin, Jun; Wong, Jiemin
2013-01-03
The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.
The MTA family proteins as novel histone H3 binding proteins
2013-01-01
Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail. PMID:23286669
Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E
2016-11-01
Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.
A brave new world of RNA-binding proteins.
Hentze, Matthias W; Castello, Alfredo; Schwarzl, Thomas; Preiss, Thomas
2018-05-01
RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein-protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA.
Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.
Miao, Yinglong; McCammon, J Andrew
2018-03-20
Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.
Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.
Grange, T; de Sa, C M; Oddos, J; Pictet, R
1987-01-01
We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805
NASA Astrophysics Data System (ADS)
Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.
2016-11-01
Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.
RNA-Binding Proteins in Female Reproductive Pathologies.
Khalaj, Kasra; Miller, Jessica E; Fenn, Christian R; Ahn, SooHyun; Luna, Rayana L; Symons, Lindsey; Monsanto, Stephany P; Koti, Madhuri; Tayade, Chandrakant
2017-06-01
RNA-binding proteins are key regulatory molecules involved primarily in post-transcriptional gene regulation of RNAs. Post-transcriptional gene regulation is critical for adequate cellular growth and survival. Recent reports have shown key interactions between these RNA-binding proteins and other regulatory elements, such as miRNAs and long noncoding RNAs, either enhancing or diminishing their response to RNA stabilization. Many RNA-binding proteins have been reported to play a functional role in mediation of cytokines involved in inflammation and immune dysfunction, and some have been classified as global post-transcriptional regulators of inflammation. The ubiquitous expression of RNA-binding proteins in a wide variety of cell types and their unique mechanisms of degradative action provide evidence that they are involved in reproductive tract pathologies. Aberrant inflammation and immune dysfunction are major contributors to the pathogenesis and disease pathophysiology of many reproductive pathologies, including ovarian and endometrial cancers in the female reproductive tract. Herein, we discuss various RNA-binding proteins and their unique contributions to female reproductive pathologies with a focus on those mediated by aberrant inflammation and immune dysfunction. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
ERIC Educational Resources Information Center
Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai
2010-01-01
The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…
Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone
Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna
2016-01-01
The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023
Hyaluronate-binding proteins of murine brain.
Marks, M S; Chi-Rosso, G; Toole, B P
1990-01-01
The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.
Clinical relevance of drug binding to plasma proteins
NASA Astrophysics Data System (ADS)
Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana
2014-12-01
Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.
Dummitt, Benjamin; Chang, Yie-Hwa
2006-06-01
Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.
Isolation and characterizations of oxalate-binding proteins in the kidney
DOE Office of Scientific and Technical Information (OSTI.GOV)
Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta
Highlights: Black-Right-Pointing-Pointer The first large-scale characterizations of oxalate-binding kidney proteins. Black-Right-Pointing-Pointer The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. Black-Right-Pointing-Pointer 38 forms of 26 unique oxalate-binding kidney proteins were identified. Black-Right-Pointing-Pointer 25/26 (96%) of identified proteins had 'L-x(3,5)-R-x(2)-[AGILPV]' domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) tomore » resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed 'L-x(3,5)-R-x(2)-[AGILPV]' as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone
Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.
2013-01-01
We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156
Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M
2016-03-14
Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.
Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.
Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur
2016-04-06
Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.
Ohno, Satoshi; Matsui, Megumi; Yokogawa, Takashi; Nakamura, Masashi; Hosoya, Takamitsu; Hiramatsu, Toshiyuki; Suzuki, Masaaki; Hayashi, Nobuhiro; Nishikawa, Kazuya
2007-03-01
An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.
The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...
Isolation of copper-binding proteins from activated sludge culture.
Fukushi, K; Kato, S; Antsuki, T; Omura, T
2001-01-01
Six copper-binding microbial proteins were isolated from activated sludge cultures grown on media containing copper at various concentrations. Molecular weights among isolated proteins were ranged from 1.3k to 1 74k dalton. Isolated proteins were compared for their copper binding capabilities. Proteins isolated from cultures grown in the presence of copper in the growth media exhibited higher copper binding capabilities than those isolated from the culture grown in the absence of copper. The highest metal uptake of 61.23 (mol copper/mol protein) was observed by a protein isolated from a culture grown with copper at a concentration of 0.25 mM. This isolated protein (CBP2) had a molecular weight of 24k dalton. Other protein exhibited copper binding capability of 4.8-32.5 (mol copper/mol protein).
Protein-protein binding before and after photo-modification of albumin
NASA Astrophysics Data System (ADS)
Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo
2016-03-01
Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.
Effect of redox partner binding on CYP101D1 conformational dynamics.
Batabyal, Dipanwita; Poulos, Thomas L
2018-06-01
We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDX D38 which forms an important ion pair with P450cam R112 at the center of the interface is Arx L39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with Arx L39 . Copyright © 2018. Published by Elsevier Inc.
Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.
He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J
2000-08-01
Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.
Sequence-Based Prediction of RNA-Binding Residues in Proteins
Walia, Rasna R.; EL-Manzalawy, Yasser; Honavar, Vasant G.; Dobbs, Drena
2017-01-01
Identifying individual residues in the interfaces of protein–RNA complexes is important for understanding the molecular determinants of protein–RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein–RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein–RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner. PMID:27787829
Characterization of binding of N'-nitrosonornicotine to protein
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hughes, M.F.
1986-01-01
The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of (/sup 14/C)NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding tomore » liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N/sub 2/ or CO:O/sub 2/ (8:2) significantly decreased the NADPH-dependent binding of (/sup 14/C)NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of (/sup 14/C)NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation.« less
The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.
Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E
2017-12-05
Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.
Lipids and lipid binding proteins: a perfect match.
Glatz, Jan F C
2015-02-01
Lipids serve a great variety of functions, ranging from structural components of biological membranes to signaling molecules affecting various cellular functions. Several of these functions are related to the unique physico-chemical properties shared by all lipid species, i.e., their hydrophobicity. The latter, however, is accompanied by a poor solubility in an aqueous environment and thus a severe limitation in the transport of lipids in aqueous compartments such as blood plasma and the cellular soluble cytoplasm. Specific proteins which can reversibly and non-covalently associate with lipids, designated as lipid binding proteins or lipid chaperones, greatly enhance the aqueous solubility of lipids and facilitate their transport between tissues and within tissue cells. Importantly, transport of lipids across biological membranes also is facilitated by specific (membrane-associated) lipid binding proteins. Together, these lipid binding proteins determine the bio-availability of their ligands, and thereby markedly influence the subsequent processing, utilization, or signaling effect of lipids. The bio-availability of specific lipid species thus is governed by the presence of specific lipid binding proteins, the affinity of these proteins for distinct lipid species, and the presence of competing ligands (including pharmaceutical compounds). Recent studies suggest that post-translational modifications of lipid binding proteins may have great impact on lipid-protein interactions. As a result, several levels of regulation exist that together determine the bio-availability of lipid species. This short review discusses the significance of lipid binding proteins and their potential application as targets for therapeutic intervention. Copyright © 2014 Elsevier Ltd. All rights reserved.
La Verde, Valentina; Dominici, Paola; Astegno, Alessandra
2018-04-30
Ca 2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca 2+ -binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca 2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca 2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca 2+ sensors is also presented.
Ice-shell purification of ice-binding proteins.
Marshall, Craig J; Basu, Koli; Davies, Peter L
2016-06-01
Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. Copyright © 2016 Elsevier Inc. All rights reserved.
Sarhan, Maen F; Van Petegem, Filip; Ahern, Christopher A
2009-11-27
Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.
Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian
2016-12-15
In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.
MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.
Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R
2016-07-08
Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.
The path to CAM6: coupled simulations with CAM5.4 and CAM5.5
NASA Astrophysics Data System (ADS)
Bogenschutz, Peter A.; Gettelman, Andrew; Hannay, Cecile; Larson, Vincent E.; Neale, Richard B.; Craig, Cheryl; Chen, Chih-Chieh
2018-01-01
This paper documents coupled simulations of two developmental versions of the Community Atmosphere Model (CAM) towards CAM6. The configuration called CAM5.4 introduces new microphysics, aerosol, and ice nucleation changes, among others to CAM. The CAM5.5 configuration represents a more radical departure, as it uses an assumed probability density function (PDF)-based unified cloud parameterization to replace the turbulence, shallow convection, and warm cloud macrophysics in CAM. This assumed PDF method has been widely used in the last decade in atmosphere-only climate simulations but has never been documented in coupled mode. Here, we compare the simulated coupled climates of CAM5.4 and CAM5.5 and compare them to the control coupled simulation produced by CAM5.3. We find that CAM5.5 has lower cloud forcing biases when compared to the control simulations. Improvements are also seen in the simulated amplitude of the Niño-3.4 index, an improved representation of the diurnal cycle of precipitation, subtropical surface wind stresses, and double Intertropical Convergence Zone biases. Degradations are seen in Amazon precipitation as well as slightly colder sea surface temperatures and thinner Arctic sea ice. Simulation of the 20th century results in a credible simulation that ends slightly colder than the control coupled simulation. The authors find this is due to aerosol indirect effects that are slightly stronger in the new version of the model and propose a solution to ameliorate this. Overall, in these early coupled simulations, CAM5.5 produces a credible climate that is appropriate for science applications and is ready for integration into the National Center for Atmospheric Research's (NCAR's) next-generation climate model.
Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.
Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura
2009-10-01
Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.
Trans‐acting translational regulatory RNA binding proteins
Harvey, Robert F.; Smith, Tom S.; Mulroney, Thomas; Queiroz, Rayner M. L.; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa
2018-01-01
The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans‐acting regulatory RNA‐binding proteins (RBPs) are necessary to provide mRNA‐specific translation, and these interact with 5′ and 3′ untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans‐acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans‐acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans‐acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: 1RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes2Translation > Translation Regulation3Translation > Translation Mechanisms PMID:29341429
Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.
Davis, J Q; Bennett, V
1993-01-01
A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.
Freed, Alexander S; Garde, Shekhar; Cramer, Steven M
2011-11-17
Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.
Korkuć, Paula; Walther, Dirk
2015-01-01
To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous
Korkuć, Paula; Walther, Dirk
2015-01-01
To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous
Binding free energy analysis of protein-protein docking model structures by evERdock.
Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio
2018-03-14
To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.
Binding free energy analysis of protein-protein docking model structures by evERdock
NASA Astrophysics Data System (ADS)
Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio
2018-03-01
To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.
Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.
Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L
1987-08-01
To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.
Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.
Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L
1987-01-01
To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded. Images PMID:2823109
Laine, Elodie; Martínez, Leandro; Blondel, Arnaud; Malliavin, Thérèse E
2010-10-06
Calmodulin (CaM) is a remarkably flexible protein which can bind multiple targets in response to changes in intracellular calcium concentration. It contains four calcium-binding sites, arranged in two globular domains. The calcium affinity of CaM N-terminal domain (N-CaM) is dramatically reduced when the complex with the edema factor (EF) of Bacillus anthracis is formed. Here, an atomic explanation for this reduced affinity is proposed through molecular dynamics simulations and free energy perturbation calculations of the EF-CaM complex starting from different crystallographic models. The simulations show that electrostatic interactions between CaM and EF disfavor the opening of N-CaM domains usually induced by calcium binding. Relative calcium affinities of the N-CaM binding sites are probed by free energy perturbation, and dissociation probabilities are evaluated with locally enhanced sampling simulations. We show that EF impairs calcium binding on N-CaM through a direct conformational restraint on Site 1, by an indirect destabilization of Site 2, and by reducing the cooperativity between the two sites. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Conformational selection in protein binding and function
Weikl, Thomas R; Paul, Fabian
2014-01-01
Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241
Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins
ERIC Educational Resources Information Center
Nixon, J. E.
1977-01-01
Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)
Mechanistic events underlying odorant binding protein chemoreception.
Golebiowski, Jérôme; Antonczak, Serge; Fiorucci, Sébastien; Cabrol-Bass, Daniel
2007-05-01
Odorant binding proteins (OBP's) are small hydrophilic proteins, belonging to the lipocalin family dedicated to bind and transport small hydrophobic ligands. Despite many works, the mechanism of ligand binding, together with the functional role of these proteins remains a topic of debate and little is known at the atomic level. The present work reports a computational study of odorants capture and release by an OBP, using both constrained and unconstrained simulations, giving a glimpse on the molecular mechanism of chemoreception. The residues at the origin of the regulation of the protein door opening are identified and a tyrosine amino-acid together with other nearby residues appear to play a crucial role in allowing this event to occur. The simulations reveal that this tyrosine and the protein's L5 loop are implicated in the ligand contact with the protein and act as an anchoring point for the ligand. The protein structural features required for the ligand entry are highly conserved among many transport proteins, suggesting that this mechanism could somewhat be extended to some members of the larger family of lipocalin. (c) 2007 Wiley-Liss, Inc.
Unconventional RNA-binding proteins: an uncharted zone in RNA biology.
Albihlal, Waleed S; Gerber, André P
2018-06-16
RNA-binding proteins play essential roles in the post-transcriptional regulation of gene expression. While hundreds of RNA-binding proteins can be predicted computationally, the recent introduction of proteome-wide approaches has dramatically expanded the repertoire of proteins interacting with RNA. Besides canonical RNA-binding proteins that contain characteristic RNA-binding domains, many proteins that lack such domains but have other well-characterised cellular functions were identified; including metabolic enzymes, heat shock proteins, kinases, as well as transcription factors and chromatin-associated proteins. In the context of these recently published RNA-protein interactome datasets obtained from yeast, nematodes, flies, plants and mammalian cells, we discuss examples for seemingly evolutionary conserved "unconventional" RNA-binding proteins that act in central carbon metabolism, stress response or regulation of transcription. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.
Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos
2017-05-13
Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.
Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony
2001-01-01
The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5′ end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (∼10-fold) in the csrA mutants. CsrB transcript stability was unaffected by csrA. The expression of a csrB-lacZ transcriptional fusion containing the region from −242 to +4 bp of the csrB gene was decreased ∼20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB. PMID:11567002
Behind the scenes of vitamin D binding protein: more than vitamin D binding.
Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M
2015-10-01
Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. Copyright © 2015 Elsevier Ltd. All rights reserved.
Multivalent DNA-binding properties of the HMG-1 proteins.
Maher, J F; Nathans, D
1996-01-01
HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884
Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana
2016-01-01
Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca2+ signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca2+ and Ca2+-CaM indicated that E2 also increases free Ca2+-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca2+-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca2+ efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca2+ signals and promote Ca2+-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca2+/CaM signals and functional linkage in the endothelial CaM target network. PMID:26987903
Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana
2016-05-13
Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
SCOWLP classification: Structural comparison and analysis of protein binding regions
Teyra, Joan; Paszkowski-Rogacz, Maciej; Anders, Gerd; Pisabarro, M Teresa
2008-01-01
Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs) might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions. The hierarchical
Trans-acting translational regulatory RNA binding proteins.
Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E
2018-05-01
The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.
Calculations of the binding affinities of protein-protein complexes with the fast multipole method
NASA Astrophysics Data System (ADS)
Kim, Bongkeun; Song, Jiming; Song, Xueyu
2010-09-01
In this paper, we used a coarse-grained model at the residue level to calculate the binding free energies of three protein-protein complexes. General formulations to calculate the electrostatic binding free energy and the van der Waals free energy are presented by solving linearized Poisson-Boltzmann equations using the boundary element method in combination with the fast multipole method. The residue level model with the fast multipole method allows us to efficiently investigate how the mutations on the active site of the protein-protein interface affect the changes in binding affinities of protein complexes. Good correlations between the calculated results and the experimental ones indicate that our model can capture the dominant contributions to the protein-protein interactions. At the same time, additional effects on protein binding due to atomic details are also discussed in the context of the limitations of such a coarse-grained model.
Concentration-dependent Cu(II) binding to prion protein
NASA Astrophysics Data System (ADS)
Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry
2008-03-01
The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.
Uchikoga, Nobuyuki; Hirokawa, Takatsugu
2010-05-11
Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles. To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG), CaM kinase kinase (CaMKK) and the plasma membrane Ca2+ ATPase pump (PMCA), and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK. The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases.
The modular architecture of protein-protein binding interfaces.
Reichmann, D; Rahat, O; Albeck, S; Meged, R; Dym, O; Schreiber, G
2005-01-04
Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.
EMSA Analysis of DNA Binding By Rgg Proteins.
LaSarre, Breah; Federle, Michael J
2013-08-20
In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).
Shorofi, Seyed Afshin
2011-11-01
This paper reports a study to examine hospitalised patients' frequency and patterns of CAM use, their reasons for CAM use, their preferences of CAMs during hospitalisation, and the association between patients' socio-demographic variables and use of each individual CAM/CAM domain. A convenience sample of 353 patients hospitalised in 19 surgical wards at four metropolitan hospitals completed a questionnaire on CAM use and socio-demographic variables. The response rate was 73.5%, and over 90% of the sample acknowledged using CAMs. Non-herbal supplements (60.3%) and massage therapy (45%) were the most frequently used CAMs, with biologically based therapies (68.8%) as well as mind-body interventions (65.4%) being the most often used CAM domains. About 1 in 10 patients (9.6%) used CAMs from all five domains. With the exception of herbal-botanical therapies, self-prayer for health reasons/spiritual healing and music therapy, all CAMs were mainly used on an 'only when needed' basis. The most common reason nominated for using CAMs was that '[it] fits into my way of life/philosophy' (26%). The majority of patients declared interest in and support for the hospital providing CAMs. Patients were most inclined to choose therapies categorised as manipulative and body-based methods (65.4%) for use in hospital. Massage therapy (53.5%) and non-herbal supplements (43.1%) were the top two CAMs favoured for use in hospital. CAM use was also dependent of socio-demographic data (age, gender, marital status, place of residence, education level, religion, and income in hospitalised patients). The use of CAMs is pervasive amongst surgical in-patients, making it feasible to initially assess these patients for CAM use and provide them with clinically approved CAMs where possible. Notwithstanding that CAM use is fairly predictable by socio-demographic variables, further studies should be directed to know the variables useful for predicting the use of each CAM approach. Copyright © 2011
21 CFR 866.5765 - Retinol-binding protein immunological test system.
Code of Federal Regulations, 2010 CFR
2010-04-01
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system...
Feldkamp, Michael D.; O'Donnell, Susan E.; Yu, Liping; Shea, Madeline A.
2010-01-01
Trifluoperazine (TFP; Stelazine™) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, 1LIN) show TFP bound to (Ca2+)4-CaM in ratios of 1, 2 or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM-kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca2+)4-CaM, and explore differential effects on the N- and C-domains of CaM, stoichiometric TFP titrations of CaM were monitored by 15N-HSQC NMR. Two TFP bound to apo CaM, while four bound to (Ca2+)4-CaM. In both cases, the preferred site was in the C-domain. During the titrations, biphasic responses for some resonances suggested inter-site interactions. TFP-binding sites in apo CaM appeared distinct from those in (Ca2+)4-CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ-motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N-domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed”, “semi-open” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca2+)4-CaM, needs to be considered a potential target of drug action. PMID:20544963
Imaging of Ep-CAM Positive Metastatic Cancer in the Lymph System
2011-01-01
bind Ep-CAM positive breast cancer cells in pH up to 8.3, which will be important for the chelating agent DOTA . The DOTA -NHS-ester is best conjugated...must be conjugated with the fluorophore (IRDye 800CW) and a chelating agent ( DOTA ), which sequesters the radio-metal completing the dual-label...done simultaneously, anti-Ep-CAM (9601) was conjugated with 5-fold excess IRDye, 5- fold excess DOTA or 500-fold excess DOTA overnight. After size
Discovering amino acid patterns on binding sites in protein complexes
Kuo, Huang-Cheng; Ong, Ping-Lin; Lin, Jung-Chang; Huang, Jen-Peng
2011-01-01
Discovering amino acid (AA) patterns on protein binding sites has recently become popular. We propose a method to discover the association relationship among AAs on binding sites. Such knowledge of binding sites is very helpful in predicting protein-protein interactions. In this paper, we focus on protein complexes which have protein-protein recognition. The association rule mining technique is used to discover geographically adjacent amino acids on a binding site of a protein complex. When mining, instead of treating all AAs of binding sites as a transaction, we geographically partition AAs of binding sites in a protein complex. AAs in a partition are treated as a transaction. For the partition process, AAs on a binding site are projected from three-dimensional to two-dimensional. And then, assisted with a circular grid, AAs on the binding site are placed into grid cells. A circular grid has ten rings: a central ring, the second ring with 6 sectors, the third ring with 12 sectors, and later rings are added to four sectors in order. As for the radius of each ring, we examined the complexes and found that 10Å is a suitable range, which can be set by the user. After placing these recognition complexes on the circular grid, we obtain mining records (i.e. transactions) from each sector. A sector is regarded as a record. Finally, we use the association rule to mine these records for frequent AA patterns. If the support of an AA pattern is larger than the predetermined minimum support (i.e. threshold), it is called a frequent pattern. With these discovered patterns, we offer the biologists a novel point of view, which will improve the prediction accuracy of protein-protein recognition. In our experiments, we produced the AA patterns by data mining. As a result, we found that arginine (arg) most frequently appears on the binding sites of two proteins in the recognition protein complexes, while cysteine (cys) appears the fewest. In addition, if we discriminate the shape
Mutation of EpCAM leads to intestinal barrier and ion transport dysfunction.
Kozan, Philip A; McGeough, Matthew D; Peña, Carla A; Mueller, James L; Barrett, Kim E; Marchelletta, Ronald R; Sivagnanam, Mamata
2015-05-01
Congenital tufting enteropathy (CTE) is a devastating diarrheal disease seen in infancy that is typically associated with villous changes and the appearance of epithelial tufts. We previously found mutations in epithelial cell adhesion molecule (EpCAM) to be causative in CTE. We developed a knock-down cell model of CTE through transfection of an EpCAM shRNA construct into T84 colonic epithelial cells to elucidate the in vitro role of EpCAM in barrier function and ion transport. Cells with EpCAM deficiency exhibited decreased electrical resistance, increased permeability, and decreased ion transport. Based on mutations in CTE patients, an in vivo mouse model was developed, with tamoxifen-inducible deletion of exon 4 in Epcam resulting in mutant protein with decreased expression. Tamoxifen treatment of Epcam (Δ4/Δ4) mice resulted in pathological features of villous atrophy and epithelial tufts, similar to those in human CTE patients, within 4 days post induction. Epcam (Δ4/Δ4) mice also showed decreased expression of tight junctional proteins, increased permeability, and decreased ion transport in the intestines. Taken together, these findings reveal mechanisms that may underlie disease in CTE. Knock-down EpCAM cell model of congenital tufting enteropathy was developed. In vivo inducible mouse model was developed resulting in mutant EpCAM protein. Cells with EpCAM deficiency demonstrated barrier and ion transport dysfunction. Tamoxifen-treated Epcam (Δ4/Δ4) mice demonstrated pathological features. Epcam (Δ4/Δ4) mice showed improper barrier function and ion transport.
Purification of Proteins Fused to Maltose-Binding Protein.
Lebendiker, Mario; Danieli, Tsafi
2017-01-01
Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.
RNA-binding proteins in plants: the tip of an iceberg?
NASA Technical Reports Server (NTRS)
Fedoroff, Nina V.; Federoff, N. V. (Principal Investigator)
2002-01-01
RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.
FK506-Binding Proteins and Their Diverse Functions.
Tong, Mingming; Jiang, Yu
2015-01-01
FK506 binding proteins (FKBPs) are a family of highly conserved proteins in eukaryotes. The prototype of this protein family, FKBP12, is the binding partner for immunosuppressive drugs FK506 and rapamycin. FKBP12 functions as a cis/trans peptidyl prolyl isomerase (PPIase) that catalyzes interconversion between prolyl cis/trans conformations. Members of the FKBP family contain one or several PPIase domains, which do not always exhibit PPIase activity yet are all essential for their function. FKBPs are involved in diverse cellular functions including protein folding, cellular signaling, apoptosis and transcription. They elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches. In this review, we provide a general summary for the structures and diverse functions of FKBPs found in mammalian cells.
CAM-related changes in chloroplastic metabolism of Mesembryanthemum crystallinum L.
Bilger, Wolfgang; Gruca, Magdalena; Mulisch, Maria; Miszalski, Zbigniew; Krupinska, Karin
2010-01-01
Crassulacean acid metabolism (CAM) is an intriguing metabolic strategy to maintain photosynthesis under conditions of closed stomata. A shift from C3 photosynthesis to CAM in Mesembryanthemum crystallinum plants was induced by high salinity (0.4 M NaCl). In CAM-performing plants, the quantum efficiencies of photosystem II and I were observed to undergo distinct diurnal fluctuations that were characterized by a strong decline at the onset of the day, midday recovery, and an evening drop. The temporal recovery of both photosystems’ efficiency at midday was associated with a more rapid induction of the electron transport rate at PSII. This recovery of the photosynthetic apparatus at midday was observed to be accompanied by extreme swelling of thylakoids. Despite these fluctuations, a persistent effect of CAM was the acceptor side limitation of PSI during the day, which was accompanied by a strongly decreased level of Rubisco protein. Diurnal changes in the efficiency of photosystems were parallel to corresponding changes in the levels of mRNAs for proteins of PSII and PSI reaction centers and for rbcL, reaching a maximum in CAM plants at midday. This might reflect a high demand for new protein synthesis at this time of the day. Hybridization of run-on transcripts with specific probes for plastid genes of M. crystallinum revealed that the changes in plastidic mRNA levels were regulated at the level of transcription. PMID:21046147
EpCAM and the biology of hepatic stem/progenitor cells
Theise, Neil D.; Schmelzer, Eva; Boulter, Luke; Gires, Olivier; van Grunsven, Leo A.
2014-01-01
Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration. PMID:25477371
Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism
Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue
2015-01-01
Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773
EMSA Analysis of DNA Binding By Rgg Proteins
LaSarre, Breah; Federle, Michael J.
2016-01-01
In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004
Conacci-Sorrell, Maralice E; Ben-Yedidia, Tamar; Shtutman, Michael; Feinstein, Elena; Einat, Paz; Ben-Ze'ev, Avri
2002-08-15
beta-catenin and plakoglobin (gamma-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. beta-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by beta-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either beta-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulated cell growth, enhanced motility, induced transformation, and produced rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression was elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreased Nr-CAM expression and antibodies to Nr-CAM inhibited the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by beta-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility.
Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A
2013-06-14
Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.
Phosphoinositide-binding proteins in autophagy.
Lystad, Alf Håkon; Simonsen, Anne
2016-08-01
Phosphoinositides represent a very small fraction of membrane phospholipids, having fast turnover rates and unique subcellular distributions, which make them perfect for initiating local temporal effects. Seven different phosphoinositide species are generated through reversible phosphorylation of the inositol ring of phosphatidylinositol (PtdIns). The negative charge generated by the phosphates provides specificity for interaction with various protein domains that commonly contain a cluster of basic residues. Examples of domains that bind phosphoinositides include PH domains, WD40 repeats, PX domains, and FYVE domains. Such domains often display specificity toward a certain species or subset of phosphoinositides. Here we will review the current literature of different phosphoinositide-binding proteins involved in autophagy. © 2016 Federation of European Biochemical Societies.
EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.
Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H; Templin, Markus F; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans
2015-01-01
Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual
Ion Binding Energies Determining Functional Transport of ClC Proteins
NASA Astrophysics Data System (ADS)
Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping
2014-06-01
The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.
Chen, Li-Ting; Liang, Wen-Xue; Chen, Shuo; Li, Ren-Ke; Tan, Jue-Ling; Xu, Peng-Fei; Luo, Liu-Fei; Wang, Lei; Yu, Shan-He; Meng, Guoyu; Li, Keqin Kathy; Liu, Ting-Xi; Chen, Zhu; Chen, Sai-Juan
2014-05-02
We previously reported a fusion protein NUP98-IQCG in an acute leukaemia, which functions as an aberrant regulator of transcriptional expression, yet the structure and function of IQCG have not been characterized. Here we use zebrafish to investigate the role of iqcg in haematopoietic development, and find that the numbers of haematopoietic stem cells and multilineage-differentiated cells are reduced in iqcg-deficient embryos. Mechanistically, IQCG binds to calmodulin (CaM) and acts as a molecule upstream of CaM-dependent kinase IV (CaMKIV). Crystal structures of complexes between CaM and IQ domain of IQCG reveal dual CaM-binding footprints in this motif, and provide a structural basis for a higher CaM-IQCG affinity when deprived of calcium. The results collectively allow us to understand IQCG-mediated calcium signalling in haematopoiesis, and propose a model in which IQCG stores CaM at low cytoplasmic calcium concentrations, and releases CaM to activate CaMKIV when calcium level rises.
Protein Binding Capacity of Different Forages Tannin
NASA Astrophysics Data System (ADS)
Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.
2018-02-01
Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.
K C, Tara Bahadur; Suga, Kanako; Isoshima, Takashi; Aigaki, Toshiro; Ito, Yoshihiro; Shiba, Kiyotaka; Uzawa, Takanori
2018-06-02
Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses. Copyright © 2018 Elsevier Inc. All rights reserved.
Integrating CAM into nursing curricula: CAM camp as an educational intervention.
Cornman, B Jane; Carr, Catherine A; Heitkemper, Margaret M
2006-05-01
In 2002, the University of Washington School of Nursing (SON) partnered with Bastyr University on a five-year plan to offer a four-week intensive "CAM Camp" (CAMp) for SON faculty members and medical students from across the country. The four-week educational program introduced attendees to various complementary and alternative medicine (CAM) modalities through didactic and experiential learning. To enhance complementary and alternative medicine content in a SON curriculum and to increase SON faculty knowledge and understanding about (1) the range of CAM therapies, (2) the theoretic and cultural backgrounds of these therapies, and (3) their potential contributions to the health of diverse populations. A descriptive pretest, posttest design was used to compare pre-CAMp CAM knowledge and CAM course content with post-CAMp knowledge levels of faculty and course CAM content. On post-CAMp surveys, familiarity with CAM modalities was rated with mixed results as compared with positive reports on the qualitative interviews. Interview results were more positive about CAM in general and were less specific about individual CAM topics. Statistically significant increases in competences were evident in each of 13 competencies rated with four competencies at P < .01. The number of required and elective courses containing CAM content increased as did the CAM content in continuing education conferences offered by the SON.
Ma, Xin; Guo, Jing; Sun, Xiao
2016-01-01
DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.
Protein interactions and ligand binding: from protein subfamilies to functional specificity.
Rausell, Antonio; Juan, David; Pazos, Florencio; Valencia, Alfonso
2010-02-02
The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as "specificity determining positions" (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating significant yet limited predictive capacity. We have systematically extended this observation to include the role of differential protein interactions in the segregation of protein subfamilies and explored in detail the structural distribution of SDPs at protein interfaces. Our results show the extensive influence of protein interactions in the evolution of protein families and the widespread association of SDPs with protein interfaces. The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.
Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T.more » maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.« less
Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.
Okada, Miki; Takezawa, Daisuke; Tachibanaki, Shuji; Kawamura, Satoru; Tokumitsu, Hiroshi; Kobayashi, Ryoji
2003-01-01
The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists. PMID:12844348
Method for estimating protein binding capacity of polymeric systems.
Sharma, Vaibhav; Blackwood, Keith A; Haddow, David; Hook, Lilian; Mason, Chris; Dye, Julian F; García-Gareta, Elena
2015-01-01
Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.
A tool for calculating binding-site residues on proteins from PDB structures.
Hu, Jing; Yan, Changhui
2009-08-03
In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB) that consists of the protein of interest and its interacting partner(s) and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. The developed tool is very useful for the research on protein binding site analysis and prediction.
Tippens, Kim; Marsman, Kevin; Zwickey, Heather
2009-04-01
Alternative medicine researchers and policy makers have classified prayer as a mind-body intervention, and thus, a modality of complementary and alternative medicine (CAM). As such, numerous epidemiological surveys of CAM utilization-which have included prayer-depict increasing CAM use, particularly in specific racial and ethnic groups. This paper discusses the implications of conflating prayer and CAM, especially regarding the definitions of both concepts and the resulting statistics of CAM utilization.
Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.
Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M
2015-01-01
Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.
Raman, E. Prabhu; MacKerell, Alexander D.
2015-01-01
The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202
ERIC Educational Resources Information Center
Meloy, Jim; And Others
1990-01-01
The relationship between computer-aided design (CAD), computer-aided manufacturing (CAM), and computer numerical control (CNC) computer applications is described. Tips for helping educate the CAM buyer on what to look for and what to avoid when searching for the most appropriate instructional CAM package are provided. (KR)
Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.
Chen, Eileen; Joseph, Simpson
2015-07-01
Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.
A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kim, Il Hwan; Pham, Van; Jablonka, Willy
Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry,more » we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.« less
A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.
Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F
2017-09-15
Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Demura, T.; Driscoll, W.J.; Lee, Y.C.
1991-01-01
Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less
Odorant-Binding Protein: Localization to Nasal Glands and Secretions
NASA Astrophysics Data System (ADS)
Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.
1986-07-01
An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.
Pestean, A; Krizbai, I; Böttcher, H; Párducz, A; Joó, F; Wolff, J R
1995-08-04
Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.
van der Vaart, Arjan
2015-05-01
Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.
Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.
Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter
2015-01-01
Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.
Marsman, Kevin; Zwickey, Heather
2009-01-01
Abstract Background Alternative medicine researchers and policy makers have classified prayer as a mind–body intervention, and thus, a modality of complementary and alternative medicine (CAM). As such, numerous epidemiological surveys of CAM utilization—which have included prayer—depict increasing CAM use, particularly in specific racial and ethnic groups. Objectives This paper discusses the implications of conflating prayer and CAM, especially regarding the definitions of both concepts and the resulting statistics of CAM utilization. PMID:19388867
Characterizing protein domain associations by Small-molecule ligand binding
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
Hanski, E; Caparon, M
1992-07-01
Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.
Phage display selection of peptides that target calcium-binding proteins.
Vetter, Stefan W
2013-01-01
Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.
Functional binding interaction identified between the axonal CAM L1 and members of the ERM family
Dickson, Tracey C.; Mintz, C. David; Benson, Deanna L.; Salton, Stephen R.J.
2002-01-01
Ayeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane–cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM–actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis. PMID:12070130
Functional binding interaction identified between the axonal CAM L1 and members of the ERM family.
Dickson, Tracey C; Mintz, C David; Benson, Deanna L; Salton, Stephen R J
2002-06-24
A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.
2017-01-01
Virtually all biological processes depend on the interaction between proteins at some point. The correct prediction of biomolecular binding free-energies has many interesting applications in both basic and applied pharmaceutical research. While recent advances in the field of molecular dynamics (MD) simulations have proven the feasibility of the calculation of protein–protein binding free energies, the large conformational freedom of proteins and complex free energy landscapes of binding processes make such calculations a difficult task. Moreover, convergence and reversibility of resulting free-energy values remain poorly described. In this work, an easy-to-use, yet robust approach for the calculation of standard-state protein–protein binding free energies using perturbed distance restraints is described. In the binding process the conformations of the proteins were restrained, as suggested earlier. Two approaches to avoid end-state problems upon release of the conformational restraints were compared. The method was evaluated by practical application to a small model complex of ubiquitin and the very flexible ubiquitin-binding domain of human DNA polymerase ι (UBM2). All computed free energy differences were closely monitored for convergence, and the calculated binding free energies had a mean unsigned deviation of only 1.4 or 2.5 kJ·mol–1 from experimental values. Statistical error estimates were in the order of thermal noise. We conclude that the presented method has promising potential for broad applicability to quantitatively describe protein–protein and various other kinds of complex formation. PMID:28898077
Odoh, Samuel O; Bondarevsky, Gary D; Karpus, Jason; Cui, Qiang; He, Chuan; Spezia, Riccardo; Gagliardi, Laura
2014-12-17
The capture of uranyl, UO2(2+), by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO2(2+) is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO2(2+) and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO2(2+) capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO2(2+) to the aqueous protein as well as of the difference between the binding free energies of UO2(2+) and other ions (Ca(2+), Cu(2+), Mg(2+), and Zn(2+)), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO2(2+) more strongly than SUP but that is also more selective for UO2(2+) over other ions. The predictions from the computations were confirmed experimentally.
Predicting protein-binding regions in RNA using nucleotide profiles and compositions.
Choi, Daesik; Park, Byungkyu; Chae, Hanju; Lee, Wook; Han, Kyungsook
2017-03-14
Motivated by the increased amount of data on protein-RNA interactions and the availability of complete genome sequences of several organisms, many computational methods have been proposed to predict binding sites in protein-RNA interactions. However, most computational methods are limited to finding RNA-binding sites in proteins instead of protein-binding sites in RNAs. Predicting protein-binding sites in RNA is more challenging than predicting RNA-binding sites in proteins. Recent computational methods for finding protein-binding sites in RNAs have several drawbacks for practical use. We developed a new support vector machine (SVM) model for predicting protein-binding regions in mRNA sequences. The model uses sequence profiles constructed from log-odds scores of mono- and di-nucleotides and nucleotide compositions. The model was evaluated by standard 10-fold cross validation, leave-one-protein-out (LOPO) cross validation and independent testing. Since actual mRNA sequences have more non-binding regions than protein-binding regions, we tested the model on several datasets with different ratios of protein-binding regions to non-binding regions. The best performance of the model was obtained in a balanced dataset of positive and negative instances. 10-fold cross validation with a balanced dataset achieved a sensitivity of 91.6%, a specificity of 92.4%, an accuracy of 92.0%, a positive predictive value (PPV) of 91.7%, a negative predictive value (NPV) of 92.3% and a Matthews correlation coefficient (MCC) of 0.840. LOPO cross validation showed a lower performance than the 10-fold cross validation, but the performance remains high (87.6% accuracy and 0.752 MCC). In testing the model on independent datasets, it achieved an accuracy of 82.2% and an MCC of 0.656. Testing of our model and other state-of-the-art methods on a same dataset showed that our model is better than the others. Sequence profiles of log-odds scores of mono- and di-nucleotides were much more powerful
NASA Astrophysics Data System (ADS)
Negi, Sunita; Rana Atilgan, Ali; Atilgan, Canan
2012-12-01
Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca+2 ion between the two lobes.
NASA Technical Reports Server (NTRS)
Yang, Tianbao; Poovaiah, B. W.
2002-01-01
We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.
Probing binding hot spots at protein-RNA recognition sites.
Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad
2016-01-29
We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Grafting odorant binding proteins on diamond bio-MEMS.
Manai, R; Scorsone, E; Rousseau, L; Ghassemi, F; Possas Abreu, M; Lissorgues, G; Tremillon, N; Ginisty, H; Arnault, J-C; Tuccori, E; Bernabei, M; Cali, K; Persaud, K C; Bergonzo, P
2014-10-15
Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins' orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications. Copyright © 2014 Elsevier B.V. All rights reserved.
A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders
Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba
2012-01-01
Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637
Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger
2017-01-01
Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570
Relationship between Hot Spot Residues and Ligand Binding Hot Spots in Protein-Protein Interfaces
Zerbe, Brandon S.; Hall, David R.
2013-01-01
In the context of protein-protein interactions, the term “hot spot” refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening. PMID:22770357
Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.
Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima
2012-08-27
In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.
Abriata, Luciano A.; Dal Peraro, Matteo
2015-01-01
Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations. PMID:26023027
Echinococcus granulosus fatty acid binding proteins subcellular localization.
Alvite, Gabriela; Esteves, Adriana
2016-05-01
Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.
NASA Technical Reports Server (NTRS)
McCormack, Kenneth J. (Inventor)
2016-01-01
A cryogenic cam butterfly valve has a body that includes an axially extending fluid conduit formed there through. A disc lug is connected to a back side of a valve disc and has a circular bore that receives and is larger than a cam of a cam shaft. The valve disc is rotatable for a quarter turn within the body about a lug axis that is offset from the shaft axis. Actuating the cam shaft in the closing rotational direction first causes the camming side of the cam of the cam shaft to rotate the disc lug and the valve disc a quarter turn from the open position to the closed position. Further actuating causes the camming side of the cam shaft to translate the valve disc into sealed contact with the valve seat. Opening rotational direction of the cam shaft reverses these motions.
Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai
2007-04-13
The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.
L1CAM in the Early Enteric and Urogenital System
Pechriggl, Elisabeth Judith; Concin, Nicole; Blumer, Michael J.; Bitsche, Mario; Zwierzina, Marit; Dudas, Jozsef; Koziel, Katarzyna; Altevogt, Peter; Zeimet, Alain-Gustave; Fritsch, Helga
2016-01-01
L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8–12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia. PMID:28026654
Yu, Kailiang; Foster, Adrianna
2016-04-01
Past studies have largely focused on hydraulic redistribution (HR) in trees, shrubs, and grasses, and recognized its role in interspecies interactions. HR in plants that conduct crassulacean acid metabolism (CAM), however, remains poorly investigated, as does the effect of HR on transpiration in different vegetation associations (i.e., tree-grass, CAM-grass, and tree-CAM associations). We have developed a mechanistic model to investigate the net direction and magnitude of HR at the patch scale for tree-grass, CAM-grass, and tree-CAM associations at the growing season to yearly timescale. The modeling results show that deep-rooted CAM plants in CAM-grass associations could perform hydraulic lift at a higher rate than trees in tree-grass associations in a relatively wet environment, as explained by a significant increase in grass transpiration rate in the shallow soil layer, balancing a lower transpiration rate by CAM plants. By comparison, trees in tree-CAM associations may perform hydraulic descent at a higher rate than those in tree-grass associations in a dry environment. Model simulations also show that hydraulic lift increases the transpiration of shallow-rooted plants, while hydraulic descent increases that of deep-rooted plants. CAM plants transpire during the night and thus perform HR during the day. Based on these model simulations, we suggest that the ability of CAM plants to perform HR at a higher rate may have different effects on the surrounding plant community than those of plants with C3 or C4 photosynthetic pathways (i.e., diurnal transpiration).
Discrete persistent-chain model for protein binding on DNA.
Lam, Pui-Man; Zhen, Yi
2011-04-01
We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society
Electrostatic contribution to the binding stability of protein-protein complexes.
Dong, Feng; Zhou, Huan-Xiang
2006-10-01
To investigate roles of electrostatic interactions in protein binding stability, electrostatic calculations were carried out on a set of 64 mutations over six protein-protein complexes. These mutations alter polar interactions across the interface and were selected for putative dominance of electrostatic contributions to the binding stability. Three protocols of implementing the Poisson-Boltzmann model were tested. In vdW4 the dielectric boundary between the protein low dielectric and the solvent high dielectric is defined as the protein van der Waals surface and the protein dielectric constant is set to 4. In SE4 and SE20, the dielectric boundary is defined as the surface of the protein interior inaccessible to a 1.4-A solvent probe, and the protein dielectric constant is set to 4 and 20, respectively. In line with earlier studies on the barnase-barstar complex, the vdW4 results on the large set of mutations showed the closest agreement with experimental data. The agreement between vdW4 and experiment supports the contention of dominant electrostatic contributions for the mutations, but their differences also suggest van der Waals and hydrophobic contributions. The results presented here will serve as a guide for future refinement in electrostatic calculation and inclusion of nonelectrostatic effects. Proteins 2006. (c) 2006 Wiley-Liss, Inc.
[Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].
Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang
2013-02-01
To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.
Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms
2015-01-01
ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114
Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael
2017-01-01
A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985
USDA-ARS?s Scientific Manuscript database
Odorant-binding proteins (OBPs) are important components in insect olfactory systems that transport semiochemicals through the aqueous sensillum lymph to surface of olfactory receptor neurons. In this study, we cloned the cDNA of odorant-binding protein 2 (BhorOBP2) in Batocera horsfieldi (Hope) and...
CXCL4 is a novel nickel-binding protein and augments nickel allergy.
Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S
2017-08-01
Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.
Binding of [51Cr]ethylenediaminetetraacetate to proteins of human plasma.
Babiker, M M
1986-01-01
Binding of [51Cr]EDTA to human plasma proteins was investigated using chemical and chromatographic techniques of separation of the proteins and protein fractions. Total plasma proteins isolated with ethanol retained 12.95 +/- 0.46% of the initial plasma activity. Proteins separated by other precipitants retained about 16% of the initial radioactivity. Globulins exhibited the highest binding capacity for [51Cr]EDTA and retained about 11.7% of the initial plasma activity following chromatographic separation. This value represents about 70% of the radioactivity bound by the total proteins of the plasma. gamma-Globulins contributed most of the binding attributed to the globulins and retained about 8.7% of the initial [51Cr]EDTA activity. The repeatedly reported underestimation of the renal glomerular filtration rate when estimated as the clearance of [51Cr]EDTA could be adequately accounted for by the extent of binding of this marker to the plasma proteins. PMID:2427701
Huang, Xiaojun; Liu, Ying; Wang, Ruiwu; Zhong, Xiaowei; Liu, Yingjie; Koop, Andrea; Chen, S. R. Wayne; Wagenknecht, Terence; Liu, Zheng
2013-01-01
Summary Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating. PMID:23868982
A dye-binding assay for measurement of the binding of Cu(II) to proteins.
Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B
2008-10-01
We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).
Ice-Binding Proteins and Their Function.
Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L
2016-06-02
Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities.
Solvent mimicry with methylene carbene to probe protein topography.
Gómez, Gabriela Elena; Monti, José Luis E; Mundo, Mariana Rocío; Delfino, José María
2015-10-06
The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding, conformational change, and interaction. This fundamental biophysical parameter is elusive in experimental measurement. Our approach to this problem relies on the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. This reagent (i) exerts solvent mimicry because its size is comparable to water and (ii) shows scant chemical selectivity because it generates extremely reactive methylene carbene. Methylation gives rise to the EM (extent of modification) signal, which is useful for scrutinizing the conformational change triggered by Ca(2+) binding to calmodulin (CaM). The increased EM observed for the full protein is dominated by the enhanced exposure of hydrophobic area in Ca(2+)-CaM. Fragmentation allowed us to quantify the methylene incorporation at specific sites. Peptide 91-106 reveals a major reorganization around the calcium 151 binding site, resulting in local ordering and a greater exposure of the hydrophobic surface. Additionally, this technique shows a high sensitivity to probe recognition between CaM and melittin (Mel). The large decrease in EM indicates the occlusion of a significant hydrophobic area upon complexation. Protection from labeling reveals a larger involvement of the N-terminal and central regions of CaM in this interaction. Despite its smaller size, Mel's differential exposure can also be quantified. Moreover, MS/MS fragmentation realizes the goal of extending the resolution of labeled sites at the amino acid level. Overall, DZN labeling emerges as a useful footprinting method capable of shedding light on physiological conformational changes and interactions.
Predicting nucleic acid binding interfaces from structural models of proteins.
Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael
2012-02-01
The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.
Predicting nucleic acid binding interfaces from structural models of proteins
Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael
2011-01-01
The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767
Brender, Jeffrey R.; Zhang, Yang
2015-01-01
The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies. PMID:26506533
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
Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.
2012-01-01
Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics. PMID:22713574
Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying
2015-01-01
Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization
A conserved NAD+ binding pocket that regulates protein-protein interactions during aging
Li, Jun; Bonkowski, Michael S.; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P.; Ling, Alvin J. Y.; Rajman, Luis A.; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L.; Steegborn, Clemens; Sinclair, David A.
2017-01-01
DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate–ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+. Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. PMID:28336669
Small Molecule Ligands of Methyl-Lysine Binding Proteins
Herold, J. Martin; Wigle, Tim J.; Norris, Jacqueline L.; Lam, Robert; Korboukh, Victoria K.; Gao, Cen; Ingerman, Lindsey A.; Kireev, Dmitri B.; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J.; Arrowsmith, Cheryl H.; Jin, Jian; Janzen, William P.; Frye, Stephen V.
2011-01-01
Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design. PMID:21417280
Membrane-Protein Binding Measured with Solution-Phase Plasmonic Nanocube Sensors
Wu, Hung-Jen; Henzie, Joel; Lin, Wan-Chen; Rhodes, Christopher; Li, Zhu; Sartorel, Elodie; Thorner, Jeremy; Yang, Peidong; Groves, Jay. T.
2013-01-01
We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed into a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrate the LSPR peak shift due to protein binding to the membrane surface and then characterize the lipid-binding specificity of a pleckstrin-homology domain protein. PMID:23085614
Characterization of the DNA binding properties of polyomavirus capsid protein
NASA Technical Reports Server (NTRS)
Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)
1993-01-01
The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.
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.
Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J
2002-09-01
gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.
Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.
Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun
2009-02-01
Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.
Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.
2008-08-19
Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to themore » lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.« less
Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation
Barros, Marilia; Nanda, Hirsh
2016-01-01
ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane
Nio, Kouki; Yamashita, Taro; Okada, Hikari; Kondo, Mitsumasa; Hayashi, Takehiro; Hara, Yasumasa; Nomura, Yoshimoto; Zeng, Sha Sha; Yoshida, Mariko; Hayashi, Tomoyuki; Sunagozaka, Hajime; Oishi, Naoki; Honda, Masao; Kaneko, Shuichi
2015-11-01
Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31
NASA Astrophysics Data System (ADS)
Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie
2016-11-01
Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.
A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.
Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A
2017-03-24
DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.
Zhang, Rong; Dzhura, Igor; Grueter, Chad E; Thiel, William; Colbran, Roger J; Anderson, Mark E
2005-09-01
L-type Ca2+ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca2+ for regulating gene transcription and vesicle secretion and triggering cell contraction. L-type Ca2+ channels include a pore-forming alpha and an auxiliary beta subunit, and alpha subunit openings are regulated by cellular Ca2+ through a mechanism involving the Ca2+-sensing protein calmodulin (CaM) and CaM binding motifs in the alpha subunit cytoplasmic C terminus. Here we show that these CaM binding motifs are "auto-agonists" that increase alpha subunit openings by binding the beta subunit. The CaM binding domains are necessary and sufficient for the alpha subunit C terminus to bind the beta subunit in vitro, and excess CaM blocks this interaction. Addition of CaM binding domains to native cardiac L-type Ca2+ channels in excised cell membrane patches increases openings, and this agonist effect is prevented by excess CaM. Recombinant LTCC openings are also increased by exogenous CaM binding domains by a mechanism requiring the beta subunit, and excess CaM blocks this effect. Thus, the bifunctional ability of the alpha subunit CaM binding motifs to competitively associate with the beta subunit or CaM provides a novel paradigm for feedback control of cellular Ca2+ entry.
New fluorescent reagents specific for Ca{sup 2+}-binding proteins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ben-Hail, Danya; Lemelson, Daniela; Israelson, Adrian
2012-09-14
Highlights: Black-Right-Pointing-Pointer New reagents specifically inhibit the activity of Ca{sup 2+}-dependent proteins. Black-Right-Pointing-Pointer FITC-Ru and EITC-Ru allow for mechanism-independent probing of Ca{sup 2+}-binding proteins. Black-Right-Pointing-Pointer Changes in reagents fluorescence allow characterization of protein Ca{sup 2+}-binding properties. -- Abstract: Ca{sup 2+} carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca{sup 2+}-dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru. These reagents were purified, characterized and found to specifically interact with andmore » markedly inhibit Ca{sup 2+}-dependent activities but not the activity of Ca{sup 2+}-independent reactions. In contrast to many reagents that serve as probes for Ca{sup 2+}, FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca{sup 2+}-binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca{sup 2+}-binding proteins, thereby facilitating better understanding of the function of Ca{sup 2+} as a key bio-regulator.« less
RNA Binding Protein-Mediated Post-Transcriptional Gene Regulation in Medulloblastoma
Bish, Rebecca; Vogel, Christine
2014-01-01
Medulloblastoma, the most common malignant brain tumor in children, is a disease whose mechanisms are now beginning to be uncovered by high-throughput studies of somatic mutations, mRNA expression patterns, and epigenetic profiles of patient tumors. One emerging theme from studies that sequenced the tumor genomes of large cohorts of medulloblastoma patients is frequent mutation of RNA binding proteins. Proteins which bind multiple RNA targets can act as master regulators of gene expression at the post-transcriptional level to co-ordinate cellular processes and alter the phenotype of the cell. Identification of the target genes of RNA binding proteins may highlight essential pathways of medulloblastomagenesis that cannot be detected by study of transcriptomics alone. Furthermore, a subset of RNA binding proteins are attractive drug targets. For example, compounds that are under development as anti-viral targets due to their ability to inhibit RNA helicases could also be tested in novel approaches to medulloblastoma therapy by targeting key RNA binding proteins. In this review, we discuss a number of RNA binding proteins, including Musashi1 (MSI1), DEAD (Asp-Glu-Ala-Asp) box helicase 3 X-linked (DDX3X), DDX31, and cell division cycle and apoptosis regulator 1 (CCAR1), which play potentially critical roles in the growth and/or maintenance of medulloblastoma. PMID:24608801
NASA Technical Reports Server (NTRS)
Yang, T.; Poovaiah, B. W.
2000-01-01
35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.
Suppavorasatit, Inthawoot; Cadwallader, Keith R
2012-08-15
The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding.
Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James
2015-12-15
Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.
Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin
2017-10-13
Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.
2015-01-01
Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein
Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R
2006-11-28
We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.
Size-dependent impact of CNTs on dynamic properties of calmodulin
NASA Astrophysics Data System (ADS)
Gao, Jian; Wang, Liming; Kang, Seung-Gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan
2014-10-01
There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca2+ regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca2+ concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still
Ruddock, L. W.; Freedman, R. B.; Klappa, P.
2000-01-01
Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding. PMID:10794419
Slaughter, Brian D.; Bieber Urbauer, Ramona J.; Urbauer, Jeffrey L.; Johnson, Carey K.
2008-01-01
Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca2+ pump. PMID:17343368
Cellulose binding domain proteins
Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.
1998-11-17
A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.
Cellulose binding domain proteins
Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy
1998-01-01
A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.
Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette
2009-12-18
Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.
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
Cooperative binding modes of Cu(II) in prion protein
NASA Astrophysics Data System (ADS)
Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry
2007-03-01
The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.
Monomeric Yeast Frataxin is an Iron-Binding Protein
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cook,J.; Bencze, K.; Jankovic, A.
Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly)more » share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less
Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N
2010-10-27
Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.
Computational design of a pH-sensitive IgG binding protein.
Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David
2014-01-14
Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.
Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma
2017-07-01
Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function, and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology, and designing enzymes with new functional capabilities. Proteins 2017; 85:1319-1335. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2.
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.
The human fatty acid-binding protein family: Evolutionary divergences and functions
2011-01-01
Fatty acid-binding proteins (FABPs) are members of the intracellular lipid-binding protein (iLBP) family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20) fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied. PMID:21504868
Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.
2016-01-01
Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661
NASA Astrophysics Data System (ADS)
Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju
2014-06-01
Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.
Exploring DNA-binding Proteins with In Vivo Chemical Cross-linking and Mass Spectrometry
Qiu, Haibo; Wang, Yinsheng
2009-01-01
DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins which are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level. PMID:19714816
Fujita, Eriko; Tanabe, Yuko; Hirose, Tomonori; Aurrand-Lions, Michel; Kasahara, Tadashi; Imhof, Beat A; Ohno, Shigeo; Momoi, Takashi
2007-12-01
IGSF4a/RA175/SynCAM (RA175) and junctional adhesion molecules (Jams) are members of the immunoglobulin superfamily with a PDZ-binding domain at their C termini. Deficiency of Ra175 (Ra175(-/-)) as well as Jam-C deficiency (Jam-C(-/-)) causes the defect of the spermatid differentiation, oligo-astheno-teratozoospermia. Ra175(-/-) elongating spermatids fail to mature further, whereas Jam-C(-/-) round spermatids lose cell polarity, and most of Jam-C(-/-) elongated spermatids are completely lost. RA175 and Jam-C seem to have similar but distinct functional roles during spermatid differentiation. Here we show that the cell polarity protein Par-3 with PDZ domains, a binding partner of Jams, is one of the associated proteins of the cytoplasmic region of RA175 in testis. Par-3 and Jam-C are partly co-localized with RA175 in the elongating and elongated spermatids; their distributions overlapped with that of RA175 on the tips of the dorsal region of the head of the elongating spermatid (steps 9 to 12) in the wild type. In the Ra175(-/-) elongating spermatid, Par-3 was absent, and Jam-C was absent or abnormally localized. The RA175 formed a ternary complex with Jam-C via interaction with Par-3. The lack of the ternary complex in the Ra175(-/-) elongating spermatid may cause the defect of the specialized adhesion structures, resulting in the oligo-astheno-teratozoospermia.
Lipid A binding proteins in macrophages detected by ligand blotting
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.
1987-05-01
Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less
Actin, actin-binding proteins, and actin-related proteins in the nucleus.
Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter
2016-04-01
Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.
Chaves, Edilânia Gomes Araújo; Weber, Simone Schneider; Báo, Sonia Nair; Pereira, Luiz Augusto; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida
2015-02-27
Despite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen. In the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides. These data indicate
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wu, R.; Wilton, R.; Cuff, M. E.
We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes butmore » have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.« less
Helledie, Torben; Jørgensen, Claus; Antonius, Marianne; Krogsdam, Ann M; Kratchmarova, Irina; Kristiansen, Karsten; Mandrup, Susanne
2002-10-01
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.
Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S
2015-12-01
Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.
Reprogramming cellular events by poly(ADP-ribose)-binding proteins
Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe
2013-01-01
Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355
Evidence for specific annexin I-binding proteins on human monocytes.
Goulding, N J; Pan, L; Wardwell, K; Guyre, V C; Guyre, P M
1996-01-01
Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions. PMID:8687405
Eberini, Ivano; Guerini Rocco, Alessandro; Ientile, Anna Rita; Baptista, António M; Gianazza, Elisabetta; Tomaselli, Simona; Molinari, Henriette; Ragona, Laura
2008-06-01
The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts
Boylan, Kristin L M; Mische, Sarah; Li, Mingang; Marqués, Guillermo; Morin, Xavier; Chia, William; Hays, Thomas S
2008-02-01
The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.
EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.
Spizzo, Gilbert; Fong, Dominic; Wurm, Martin; Ensinger, Christian; Obrist, Peter; Hofer, Carina; Mazzoleni, Guido; Gastl, Guenther; Went, Philip
2011-05-01
Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term 'EpCAM overexpression' was reserved for tissues showing a total immunostaining score >4. EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.
Identifying DNA-binding proteins using structural motifs and the electrostatic potential
Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.
2004-01-01
Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290
Plant ice-binding (antifreeze) proteins
USDA-ARS?s Scientific Manuscript database
Proteins that determine the temperature at which ice crystals will form in water-based solutions in cells and tissues, that bind to growing ice crystals, thus affecting their size, and that impact ice re-crystallization have been widely-documented and studied in many plant, bacterial, fungal, insect...
Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I
1988-11-25
Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group
Accurate and sensitive quantification of protein-DNA binding affinity.
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
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
2010-01-01
Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600
Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins
ERIC Educational Resources Information Center
Ingersoll, Christine M.; Strollo, Christen M.
2007-01-01
The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.
The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).
Ganesan, Lakshmi; Buchwald, Peter
2013-04-01
The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.
The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)
Ganesan, Lakshmi; Buchwald, Peter
2013-01-01
The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742
Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A
2015-01-01
For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Finding the target sites of RNA-binding proteins
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
Definition of IgG- and albumin-binding regions of streptococcal protein G.
Akerström, B; Nielsen, E; Björck, L
1987-10-05
Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.
RNA binding properties of the US11 protein from four primate simplexviruses.
Tohme, Sarah; Cukier, Cyprian D; Severini, Alberto
2011-11-03
The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.
RNA binding properties of the US11 protein from four primate simplexviruses
2011-01-01
Background The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. Methods We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. Results and Conclusions The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins. PMID:22054255
Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.
Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per
2015-08-04
Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.
RNA–protein binding interface in the telomerase ribonucleoprotein
Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.
2011-01-01
Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986
Abnormal placental development and early embryonic lethality in EpCAM-null mice.
Nagao, Keisuke; Zhu, Jianjian; Heneghan, Mallorie B; Hanson, Jeffrey C; Morasso, Maria I; Tessarollo, Lino; Mackem, Susan; Udey, Mark C
2009-12-31
EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs.
Preorganization of molecular binding sites in designed diiron proteins.
Maglio, Ornella; Nastri, Flavia; Pavone, Vincenzo; Lombardi, Angela; DeGrado, William F
2003-04-01
De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (K(diss) = 0.001 fM), but the dissociation constant increases to 0.6 nM (deltadeltaG = 5.4 kcalmol monomer) as the active-site cavity is increased to accommodate small molecules.
Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites
Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.
2015-01-01
Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507
Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography
NASA Technical Reports Server (NTRS)
Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.
2000-01-01
Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.
Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K
2000-10-01
Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.
Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J
2016-05-01
Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
Comparative studies of human and chicken retinol-binding proteins and prealbumins.
Kopelman, M; Mokady, S; Cogan, U
1976-08-09
Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.
Ribosome protection by antibiotic resistance ATP-binding cassette protein.
Su, Weixin; Kumar, Veerendra; Ding, Yichen; Ero, Rya; Serra, Aida; Lee, Benjamin Sian Teck; Wong, Andrew See Weng; Shi, Jian; Sze, Siu Kwan; Yang, Liang; Gao, Yong-Gui
2018-05-15
The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins. Copyright © 2018 the Author(s). Published by PNAS.
Pacini, Stefania; Punzi, Tiziana; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco
2012-01-01
Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential.
Pinne, Marija; Matsunaga, James; Haake, David A
2012-11-01
Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.
Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe
2015-06-01
Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.
Identification of DNA-Binding Proteins Using Structural, Electrostatic and Evolutionary Features
Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir
2009-01-01
Summary DNA binding proteins (DBPs) often take part in various crucial processes of the cell's life cycle. Therefore, the identification and characterization of these proteins are of great importance. We present here a random forests classifier for identifying DBPs among proteins with known three-dimensional structures. First, clusters of evolutionarily conserved regions (patches) on the protein's surface are detected using the PatchFinder algorithm; previous studies showed that these regions are typically the proteins' functionally important regions. Next, we train a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein including its dipole moment. Using 10-fold cross validation on a dataset of 138 DNA-binding proteins and 110 proteins which do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of previously published methods. Furthermore, when we tested 5 different methods on 11 new DBPs which did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA. PMID:19233205
Evans, T. Idil Apak; Hell, Johannes; Shea, Madeline A.
2011-01-01
Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca2+ channel (CaV1.2) regulates Ca2+ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with CaV1.2 under low resting [Ca2+], but is poised to change conformation and position when intracellular [Ca2+] rises. CaM binding Ca2+, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A1588, and C1614 and the IQ motif studied as overlapping peptides IQ1644 and IQ′1650 as well as their effect on calcium binding. (Ca2+)4-CaM bound to all four peptides very favorably (Kd ≤ 2 nM). Linkage analysis showed that IQ1644–1670 bound with a Kd ~1 pM. In the pre-IQ region, (Ca2+)2-N-domain bound preferentially to A1588, while (Ca2+)2-C-domain preferred C1614. When bound to C1614, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM. PMID:21757287
Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.
Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub
2017-01-01
Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.
Rational redesign of neutral endopeptidase binding to merlin and moesin proteins
Niv, Masha Y; Iida, Katsuyuki; Zheng, Rong; Horiguchi, Akio; Shen, Ruoqian; Nanus, David M
2009-01-01
Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K19K20K21 basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E159Q160 in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N159L160 residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N159L160Y161K162M163 in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function. PMID:19388049
Metal binding proteins, recombinant host cells and methods
Summers, Anne O.; Caguiat, Jonathan J.
2004-06-15
The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sampaio, S.O.; Mei, C.; Butcher, E.C.
The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type I transmembrane adhesion molecule comprising two distal Ig domains involved in {alpha}4{beta}7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal Ig domain homologous to IgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three Ig domains are encoded by a distinct exon, whereasmore » the transmembrane, cytoplasmic tail, and 3{prime}-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third Ig domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/IgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support {alpha}4{beta}7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5{prime} of the transcription start site include tandem nuclear factor-KB sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs. {alpha}4{beta}7-dependent adhesive events in lymphocyte trafficking. 46 refs., 6 figs., 1 tab.« less
Enrichment, Isolation and Molecular Characterization of EpCAM-Negative Circulating Tumor Cells.
Lampignano, Rita; Schneck, Helen; Neumann, Martin; Fehm, Tanja; Neubauer, Hans
2017-01-01
The presence of EpCAM-positive circulating tumor cells (CTCs) in the peripheral blood is associated with poor clinical outcomes in breast, colorectal and prostate cancer, as well as the prognosis of other tumor types. In addition, recent studies have suggested that the presence of CTCs undergoing epithelial-to-mesenchymal transition and, as such, may exhibit reduced or no expression of epithelial proteins e.g. EpCAM, might be related to disease progression in metastatic breast cancer (MBC) patients. Analyzing the neoplastic nature of this EpCAM-low/negative (EpCAM-neg) subpopulation remains an open issue as the current standard detection methods for CTCs are not efficient at identifying this subpopulation of cells. The possible association of EpCAM-neg CTCs with EpCAM-positive (EpCAM-pos) CTCs and role in the clinicopathological features and prognosis of MBC patients has still to be demonstrated. Several technologies have been developed and are currently being tested for the identification and the downstream analyses of EpCAM-pos CTCs. These technologies can be adapted and implemented into workflows to isolate and investigate EpCAM-neg cells to understand their biology and clinical relevance. This chapter will endeavour to explain the rationale behind the identification and analyses of all CTC subgroups, as well as to review the current strategies employed to enrich, isolate and characterize EpCAM-negative CTCs. Finally, the latest findings in the field will briefly be discussed with regard to their clinical relevance.
Gong, Xinqi; Wang, Panwen; Yang, Feng; Chang, Shan; Liu, Bin; He, Hongqiu; Cao, Libin; Xu, Xianjin; Li, Chunhua; Chen, Weizu; Wang, Cunxin
2010-11-15
Protein-protein docking has made much progress in recent years, but challenges still exist. Here we present the application of our docking approach HoDock in CAPRI. In this approach, a binding site prediction is implemented to reduce docking sampling space and filter out unreasonable docked structures, and a network-based enhanced combinatorial scoring function HPNCscore is used to evaluate the decoys. The experimental information was combined with the predicted binding site to pick out the most likely key binding site residues. We applied the HoDock method in the recent rounds of the CAPRI experiments, and got good results as predictors on targets 39, 40, and 41. We also got good results as scorers on targets 35, 37, 40, and 41. This indicates that our docking approach can contribute to the progress of protein-protein docking methods and to the understanding of the mechanism of protein-protein interactions. © 2010 Wiley-Liss, Inc.
HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features.
Zaman, Rianon; Chowdhury, Shahana Yasmin; Rashid, Mahmood A; Sharma, Alok; Dehzangi, Abdollah; Shatabda, Swakkhar
2017-01-01
DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM) as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.
Wang, Wei; Liu, Juan; Sun, Lin
2016-07-01
Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Binding ligand prediction for proteins using partial matching of local surface patches.
Sael, Lee; Kihara, Daisuke
2010-01-01
Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.
Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches
Sael, Lee; Kihara, Daisuke
2010-01-01
Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group. PMID:21614188
1984-01-01
By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal
Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.
Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna
2012-03-01
Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.
A Crayfish Insulin-like-binding Protein
Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir
2013-01-01
Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079
Update of the ATTRACT force field for the prediction of protein-protein binding affinity.
Chéron, Jean-Baptiste; Zacharias, Martin; Antonczak, Serge; Fiorucci, Sébastien
2017-06-05
Determining the protein-protein interactions is still a major challenge for molecular biology. Docking protocols has come of age in predicting the structure of macromolecular complexes. However, they still lack accuracy to estimate the binding affinities, the thermodynamic quantity that drives the formation of a complex. Here, an updated version of the protein-protein ATTRACT force field aiming at predicting experimental binding affinities is reported. It has been designed on a dataset of 218 protein-protein complexes. The correlation between the experimental and predicted affinities reaches 0.6, outperforming most of the available protocols. Focusing on a subset of rigid and flexible complexes, the performance raises to 0.76 and 0.69, respectively. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Volatile anesthetic binding to proteins is influenced by solvent and aliphatic residues.
Streiff, John H; Jones, Keith A
2008-10-01
The main objective of this work was to characterize VA binding sites in multiple anesthetic target proteins. A computational algorithm was used to quantify the solvent exclusion and aliphatic character of amphiphilic pockets in the structures of VA binding proteins. VA binding sites in the protein structures were defined as the pockets with solvent exclusion and aliphatic character that exceeded minimum values observed in the VA binding sites of serum albumin, firefly luciferase, and apoferritin. We found that the structures of VA binding proteins are enriched in these pockets and that the predicted binding sites were consistent with experimental determined binding locations in several proteins. Autodock3 was used to dock the simulated molecules of 1,1,1,2,2-pentafluoroethane, difluoromethyl 1,1,1,2-tetrafluoroethyl ether, and sevoflurane and the isomers of halothane and isoflurane into these potential binding sites. We found that the binding of the various VA molecules to the amphiphilic pockets is driven primarily by VDW interactions and to a lesser extent by weak hydrogen bonding and electrostatic interactions. In addition, the trend in Delta G binding values follows the Meyer-Overton rule. These results suggest that VA potencies are related to the VDW interactions between the VA ligand and protein target. It is likely that VA bind to sites with a high degree of solvent exclusion and aliphatic character because aliphatic residues provide favorable VDW contacts and weak hydrogen bond donors. Water molecules occupying these sites maintain pocket integrity, associate with the VA ligand, and diminish the unfavorable solvation enthalpy of the VA. Water molecules displaced into the bulk by the VA ligand may provide an additional favorable enthalpic contribution to VA binding. Anesthesia is a component of many health related procedures, the outcomes of which could be improved with a better understanding of the molecular targets and mechanisms of anesthetic action.
Expression and affinity purification of recombinant proteins from plants
NASA Technical Reports Server (NTRS)
Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun
2002-01-01
With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).
The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.
Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng
2009-12-01
Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.
Consequences of systematic model drift in DYNAMO MJO hindcasts with SP-CAM and CAM5
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hannah, Walter M.; Maloney, Eric D.; Pritchard, Michael S.
Hindcast simulations of MJO events during the dynamics of the MJO (DYNAMO) field campaign are conducted with two models, one with conventional parameterization (CAM5) and a comparable model that utilizes superparameterization (SP–CAM). SP–CAM is shown to produce a qualitatively better reproduction of the fluctuations of precipitation and low–level zonal wind associated with the first two DYNAMO MJO events compared to CAM5. Interestingly, skill metrics using the real–time multivariate MJO index (RMM) suggest the opposite conclusion that CAM5 has more skill than SP–CAM. This inconsistency can be explained by a systematic increase of RMM amplitude with lead time, which results frommore » a drift of the large–scale wind field in SP–CAM that projects strongly onto the RMM index. CAM5 hindcasts exhibit a contraction of the moisture distribution, in which extreme wet and dry conditions become less frequent with lead time. SP–CAM hindcasts better reproduce the observed moisture distribution, but also have stronger drift patterns of moisture budget terms, such as an increase in drying by meridional advection in SP–CAM. This advection tendency in SP–CAM appears to be associated with enhanced off–equatorial synoptic eddy activity with lead time. In conclusion, systematic drift moisture tendencies in SP–CAM are of similar magnitude to intraseasonal moisture tendencies, and therefore are important for understanding MJO prediction skill.« less
Review: Lutheran/B-CAM: a laminin receptor on red blood cells and in various tissues.
Kikkawa, Yamato; Miner, Jeffrey H
2005-01-01
The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is a transmembrane receptor with five immunoglobulin-like domains in its extracellular region; it is therefore classified as a member of the immunoglobulin (Ig) gene family. Lu/B-CAM is observed not only on red blood cells, but also on a subset of muscle and epithelial cells in various tissues. Recently, several groups have reported that Lu/B-CAM is a novel receptor for laminin a5. The laminin a5 chain is a component of the laminin-511 (alpha 5 beta 1 gamma 1), -521 (alpha 5 beta 2 gamma 1), and -523 (alpha 5 beta 2 gamma 3) heterotrimers and is expressed throughout the mammalian body. We also have shown that Lu/B-CAM is co-localized with laminin alpha 5 in various tissues. Although the biological role of Lu/B-CAM remains unclear, the specific binding of Lu/B-CAM to laminin alpha 5 suggests that it plays an important role in developmental and physiological processes. It also is necessary to investigate further the interaction between Lu/B-CAM and laminin a5 in pathological processes, including sickle cell disease and cancer.
Sinars, Cindy R.; Cheung-Flynn, Joyce; Rimerman, Ronald A.; Scammell, Jonathan G.; Smith, David F.; Clardy, Jon
2003-01-01
The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex. PMID:12538866
A versatile assay for RNA-binding proteins in living cells
Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W.; Castello, Alfredo
2014-01-01
RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein–mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology. PMID:24664470
Winter, Klaus; Holtum, Joseph A M
2014-07-01
Facultative crassulacean acid metabolism (CAM) describes the optional use of CAM photosynthesis, typically under conditions of drought stress, in plants that otherwise employ C3 or C4 photosynthesis. In its cleanest form, the upregulation of CAM is fully reversible upon removal of stress. Reversibility distinguishes facultative CAM from ontogenetically programmed unidirectional C3-to-CAM shifts inherent in constitutive CAM plants. Using mainly measurements of 24h CO2 exchange, defining features of facultative CAM are highlighted in five terrestrial species, Clusia pratensis, Calandrinia polyandra, Mesembryanthemum crystallinum, Portulaca oleracea and Talinum triangulare. For these, we provide detailed chronologies of the shifts between photosynthetic modes and comment on their usefulness as experimental systems. Photosynthetic flexibility is also reviewed in an aquatic CAM plant, Isoetes howellii. Through comparisons of C3 and CAM states in facultative CAM species, many fundamental biochemical principles of the CAM pathway have been uncovered. Facultative CAM species will be of even greater relevance now that new sequencing technologies facilitate the mapping of genomes and tracking of the expression patterns of multiple genes. These technologies and facultative CAM systems, when joined, are expected to contribute in a major way towards our goal of understanding the essence of CAM. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela
2009-12-25
{sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUPmore » is able to adapt to allow for many successful binding partners.« less
Marsh, Lorraine
2015-01-01
Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.
USDA-ARS?s Scientific Manuscript database
Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...
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.
Computational Design of Ligand Binding Proteins with High Affinity and Selectivity
Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W.; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G.; Johnsson, Kai; Stoddard, Barry L.; Baker, David
2014-01-01
The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics. PMID:24005320
NASA Astrophysics Data System (ADS)
Vijaykumar, Adithya; ten Wolde, Pieter Rein; Bolhuis, Peter G.
2018-03-01
To predict the response of a biochemical system, knowledge of the intrinsic and effective rate constants of proteins is crucial. The experimentally accessible effective rate constant for association can be decomposed in a diffusion-limited rate at which proteins come into contact and an intrinsic association rate at which the proteins in contact truly bind. Reversely, when dissociating, bound proteins first separate into a contact pair with an intrinsic dissociation rate, before moving away by diffusion. While microscopic expressions exist that enable the calculation of the intrinsic and effective rate constants by conducting a single rare event simulation of the protein dissociation reaction, these expressions are only valid when the substrate has just one binding site. If the substrate has multiple binding sites, a bound enzyme can, besides dissociating into the bulk, also hop to another binding site. Calculating transition rate constants between multiple states with forward flux sampling requires a generalized rate expression. We present this expression here and use it to derive explicit expressions for all intrinsic and effective rate constants involving binding to multiple states, including rebinding. We illustrate our approach by computing the intrinsic and effective association, dissociation, and hopping rate constants for a system in which a patchy particle model enzyme binds to a substrate with two binding sites. We find that these rate constants increase as a function of the rotational diffusion constant of the particles. The hopping rate constant decreases as a function of the distance between the binding sites. Finally, we find that blocking one of the binding sites enhances both association and dissociation rate constants. Our approach and results are important for understanding and modeling association reactions in enzyme-substrate systems and other patchy particle systems and open the way for large multiscale simulations of such systems.
Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei
NASA Technical Reports Server (NTRS)
Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.
1993-01-01
Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.
Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins
Ardi, Veronica C.; Alexander, Leslie D.; Johnson, Victoria; McAlpine, Shelli R.
2011-01-01
Heat shock protein 90 (Hsp90) accounts for 1–2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is over-expressed (3–6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90’s ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90’s MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins. PMID:21950602
A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.
Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San
2016-01-01
Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.
Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.
Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G
2016-01-01
While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.
NASA Astrophysics Data System (ADS)
Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.
2016-01-01
The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.
Stern, M; Gellermann, B; Wieser, H
1990-10-01
To investigate postnatal maturational profiles of functional and biochemical properties of rat small intestinal microvillus membranes (MVM), we did a longitudinal study in rats from birth to the age of 12 weeks. In parallel, we studied binding of cow's milk proteins and of the wheat gliadin peptide B 3142, as well as MVM proteins (SDS-PAGE). Changes in MVM fluidity and lipid composition exhibited early (0-4 weeks) and intermediate and late (6-12 weeks) patterns, as has been published earlier. Postnatal changes of food protein and peptide binding occurred early during the observation period, not related to weaning. There was not much further change in binding after 6-8 weeks. Developmental profiles of MVM protein and some lipid changes resembled, but did not equal, changes in food protein binding. We conclude that changes in MVM biochemical composition affect MVM binding characteristics. In particular, high molecular weight MVM proteins (susceptible to trypsin treatment) appear to play a role in postnatal maturational differences in MVM food protein binding.
Gunasekaran, Vinoth Prasanna; Nishi, Kumari; Sivakumar, Dakshinamurthy; Sivaraman, Thirunavukkarasu; Mathan, Ganeshan
2018-04-30
In spite of advances in breast cancer treatment and early diagnosis, drug toxicity, cancer relapse, multidrug resistance and metastasis are the major impediment to the developments of efficient drugs. However, unique druggable targets of cancer cells distinct from the normal cells provide new rationale in cancer treatment. Previous reports clearly emphasize the differential expression and localization of Y box binding protein-1 (YB-1) between normal breast tissues and different stages of breast cancer. Y box binding protein-1 is DNA as well as RNA binding protein involved in transcription and translation regulation of various proteins involved in cancer progression, apoptosis, cell cycle, epithelial to mesenchymal transition (EMT) and drug resistance. Particularly, during doxorubicin (DOX) treatment and cancer relapse conditions, YB-1 expression was very high in breast cancer tissues and localized in to nucleus which further favours DOX efflux and metastasis. Moreover, siRNA mediated silencing of YB-1 reduces breast cancer progression and metastasis. In this rationale, using an array of computational methods, 2,4-dihydroxy-5-pyrimidinyl imidothiocarbomate (DPI) has been screened out as a drug-likeness antagonist to the YB-1for cancer treatment. In this study, we determined that DPI was toxic to breast cancer cell lines as individual drug as well as in combination with DOX. Moreover, immunofluorescence and confocal studies showed that DPI decreases DOX induced YB-1 nuclear translocation and increases DOX accumulation in breast cancer cell line. A G1/G0 phase cell cycle arrest and apoptosis was also induced by DPI. Moreover, DPI modulated YB-1 downstream targets such as p53, caspase-3, CDK-1 which are involved in cell cycle progression and apoptosis. Further, metastatic functional analysis revealed that DPI inhibits cell adhesion, migration, invasion in aggressive metastatic cell line and inhibits angiogenesis in chick embryonic chorioallantoic membrane (CAM) model
Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.
Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael
2013-01-01
Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor
Size-dependent impact of CNTs on dynamic properties of calmodulin.
Gao, Jian; Wang, Liming; Kang, Seung-gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan
2014-11-07
There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca(2+)-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca(2+)-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca(2+)-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca(2+) regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca(2+) concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.
Fujita, Eriko; Tanabe, Yuko; Hirose, Tomonori; Aurrand-Lions, Michel; Kasahara, Tadashi; Imhof, Beat A.; Ohno, Shigeo; Momoi, Takashi
2007-01-01
IGSF4a/RA175/SynCAM (RA175) and junctional adhesion molecules (Jams) are members of the immunoglobulin superfamily with a PDZ-binding domain at their C termini. Deficiency of Ra175 (Ra175−/−) as well as Jam-C deficiency (Jam-C−/−) causes the defect of the spermatid differentiation, oligo-astheno-teratozoospermia. Ra175−/− elongating spermatids fail to mature further, whereas Jam-C−/− round spermatids lose cell polarity, and most of Jam-C−/− elongated spermatids are completely lost. RA175 and Jam-C seem to have similar but distinct functional roles during spermatid differentiation. Here we show that the cell polarity protein Par-3 with PDZ domains, a binding partner of Jams, is one of the associated proteins of the cytoplasmic region of RA175 in testis. Par-3 and Jam-C are partly co-localized with RA175 in the elongating and elongated spermatids; their distributions overlapped with that of RA175 on the tips of the dorsal region of the head of the elongating spermatid (steps 9 to 12) in the wild type. In the Ra175−/− elongating spermatid, Par-3 was absent, and Jam-C was absent or abnormally localized. The RA175 formed a ternary complex with Jam-C via interaction with Par-3. The lack of the ternary complex in the Ra175−/− elongating spermatid may cause the defect of the specialized adhesion structures, resulting in the oligo-astheno-teratozoospermia. PMID:18055550
RNA–protein binding kinetics in an automated microfluidic reactor
Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.
2009-01-01
Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214
Li, Pei; Zhang, Jing; Zhu, Yuanfang; Liu, Ming; Xuan, Jin
2015-11-01
Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene. We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction. With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300. This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.
Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M
2018-06-01
Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.
Kinetic rate constant prediction supports the conformational selection mechanism of protein binding.
Moal, Iain H; Bates, Paul A
2012-01-01
The prediction of protein-protein kinetic rate constants provides a fundamental test of our understanding of molecular recognition, and will play an important role in the modeling of complex biological systems. In this paper, a feature selection and regression algorithm is applied to mine a large set of molecular descriptors and construct simple models for association and dissociation rate constants using empirical data. Using separate test data for validation, the predicted rate constants can be combined to calculate binding affinity with accuracy matching that of state of the art empirical free energy functions. The models show that the rate of association is linearly related to the proportion of unbound proteins in the bound conformational ensemble relative to the unbound conformational ensemble, indicating that the binding partners must adopt a geometry near to that of the bound prior to binding. Mirroring the conformational selection and population shift mechanism of protein binding, the models provide a strong separate line of evidence for the preponderance of this mechanism in protein-protein binding, complementing structural and theoretical studies.
Alspach, Elise; Stewart, Sheila A.
2016-01-01
Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014). PMID:27453911
Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.
de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy
2016-11-01
Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p < 0.05). When epididymal sperm were treated with milk followed by seminal plasma, coating of sperm with BSP proteins was not significantly reduced (57.6 %; p > 0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.
Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment
Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert
2016-01-01
In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context. PMID:27668217
SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G
Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus
2017-01-01
The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063
Lead(II) Binding in Natural and Artificial Proteins
Cangelosi, Virginia; Ruckthong, Leela; Pecoraro, Vincent L.
2017-01-01
This article describes recent attempts to understand the biological chemistry of lead using a synthetic biology approach. Lead binds to a variety of different biomolecules ranging from enzymes and regulatory and signaling proteins to bone matrix. We have focused on the interactions of this element in thiolate-rich sites that are found in metalloregulatory proteins such as Pbr, Znt, and CadC and in enzymes such as δ-aminolevulinic acid dehydratase (ALAD). In these proteins, Pb(II) is often found as a homoleptic and hemidirectic Pb(II)(SR)3− complex. Using first principles of biophysics, we have developed relatively short peptides that can associate into three-stranded coiled coils (3SCCs), in which a cysteine group is incorporated into the hydrophobic core to generate a (cysteine)3 binding site. We describe how lead may be sequestered into these sites, the characteristic spectral features may be observed for such systems and we provide crystallographic insight on metal binding. The Pb(II)(SR)3− that is revealed within these α-helical assemblies forms a trigonal pyramidal structure (having an endo orientation) with distinct conformations than are also found in natural proteins (having an exo conformation). This structural insight, combined with 207Pb NMR spectroscopy, suggests that while Pb(II) prefers hemidirected Pb(II)(SR)3− scaffolds regardless of the protein fold, the way this is achieved within α-helical systems is different than in β-sheet or loop regions of proteins. These interactions between metal coordination preference and protein structural preference undoubtedly are exploited in natural systems to allow for protein conformation changes that define function. Thus, using a design approach that separates the numerous factors that lead to stable natural proteins allows us to extract fundamental concepts on how metals behave in biological systems. PMID:28731303
Feng, Rui; Xu, Jianjun; Minobe, Etsuko; Kameyama, Asako; Yang, Lei; Yu, Lifeng; Hao, Liying; Kameyama, Masaki
2014-05-01
The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.
Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1
Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.
1989-01-01
Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603
Cam-Operated Pitch-Change Apparatus
NASA Technical Reports Server (NTRS)
Barnes, P. E. (Inventor)
1978-01-01
A pitch-change apparatus for a ducted thrust fan having a plurality of variable pitch blades employs a camming ring mounted coaxially at the hub at an axially fixed station along the hub axis for rotation about the hub axis both with the blades and relative to the blades. The ring has a generally spherical outer periphery and a plurality of helical camming grooves extending in a generally spherical plane on the periphery. Each of the variable pitch blades is connected to a pitch-change horn having a cam follower mounted on its outer end, and the camming ring and the horns are so arranged about the hub axis that the plurality of followers on the horns engage respectively the plurality of helical camming grooves. Rotary drive means rotates the camming ring relative to the blades to cause blade pitch to be changed through the cooperative operation of the camming grooves on the ring and the cam followers on the pitch-change horns.
A Novel Kinesin-Like Protein with a Calmodulin-Binding Domain
NASA Technical Reports Server (NTRS)
Wang, W.; Takezawa, D.; Narasimhulu, S. B.; Reddy, A. S. N.; Poovaiah, B. W.
1996-01-01
Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with S-35-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCKI is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca(2+)/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.
Structural Basis for Activation of Fatty Acid-binding Protein 4
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gillilan,R.; Ayers, S.; Noy, N.
2007-01-01
Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPAR{gamma} in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicatesmore » that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.« less
PepComposer: computational design of peptides binding to a given protein surface
Obarska-Kosinska, Agnieszka; Iacoangeli, Alfredo; Lepore, Rosalba; Tramontano, Anna
2016-01-01
There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver. PMID:27131789
Why are hyperactive ice-binding-proteins so active?
NASA Astrophysics Data System (ADS)
Braslavsky, Ido; Celik, Yeliz; Pertaya, Natalya; Eun Choi, Young; Bar, Maya; Davies, Peter L.
2008-03-01
Ice binding proteins (IBPs), also called `antifreeze proteins' or `ice structuring proteins', are a class of proteins that protect organisms from freezing injury. These proteins have many applications in medicine and agriculture, and as a platform for future biotechnology applications. One of the interesting questions in this field focuses on the hyperactivity of some IBPs. Ice binding proteins can be classified in two groups: moderate ones that can depress the freezing point up to ˜1.0 ^oC and hyperactive ones that can depress the freezing point several-fold further even at lower concentrations. It has been suggested that the hyperactivity of IBPs stem from the fact that they block growth out of specific ice surfaces, more specifically the basal planes of ice. Here we show experimental results based on fluorescence microscopy, highlighting the differences between moderate IBPs and hyperactive IBPs. These include direct evidence for basal plane affinity of hyperactive IBPs, the effects of IBPs on growth-melt behavior of ice and the dynamics of their interaction with ice.
Pedersen, S A; Kristiansen, E; Andersen, R A; Zachariassen, K E
2007-04-01
The effect of cadmium (Cd) exposure on Cd-binding ligands was investigated for the first time in a beetle (Coleoptera), using the mealworm Tenebrio molitor (L) as a model species. Exposure to Cd resulted in an approximate doubling of the Cd-binding capacity of the protein extracts from whole animals. Analysis showed that the increase was mainly explained by the induction of a Cd-binding protein of 7134.5 Da, with non-metallothionein characteristics. Amino acid analysis and de novo sequencing revealed that the protein has an unusually high content of the acidic amino acids aspartic and glutamic acid that may explain how this protein can bind Cd even without cysteine residues. Similarities in the amino acid composition suggest it to belong to a group of little studied proteins often referred to as "Cd-binding proteins without high cysteine content". This is the first report on isolation and peptide sequence determination of such a protein from a coleopteran.
Park, Kellie A; Ribic, Adema; Laage Gaupp, Fabian M; Coman, Daniel; Huang, Yuegao; Dulla, Chris G; Hyder, Fahmeed; Biederer, Thomas
2016-07-13
Select adhesion proteins control the development of synapses and modulate their structural and functional properties. Despite these important roles, the extent to which different synapse-organizing mechanisms act across brain regions to establish connectivity and regulate network properties is incompletely understood. Further, their functional roles in different neuronal populations remain to be defined. Here, we applied diffusion tensor imaging (DTI), a modality of magnetic resonance imaging (MRI), to map connectivity changes in knock-out (KO) mice lacking the synaptogenic cell adhesion protein SynCAM 1. This identified reduced fractional anisotropy in the hippocampal CA3 area in absence of SynCAM 1. In agreement, mossy fiber refinement in CA3 was impaired in SynCAM 1 KO mice. Mossy fibers make excitatory inputs onto postsynaptic specializations of CA3 pyramidal neurons termed thorny excrescences and these structures were smaller in the absence of SynCAM 1. However, the most prevalent targets of mossy fibers are GABAergic interneurons and SynCAM 1 loss unexpectedly reduced the number of excitatory terminals onto parvalbumin (PV)-positive interneurons in CA3. SynCAM 1 KO mice additionally exhibited lower postsynaptic GluA1 expression in these PV-positive interneurons. These synaptic imbalances in SynCAM 1 KO mice resulted in CA3 disinhibition, in agreement with reduced feedforward inhibition in this network in the absence of SynCAM 1-dependent excitatory drive onto interneurons. In turn, mice lacking SynCAM 1 were impaired in memory tasks involving CA3. Our results support that SynCAM 1 modulates excitatory mossy fiber inputs onto both interneurons and principal neurons in the hippocampal CA3 area to balance network excitability. This study advances our understanding of synapse-organizing mechanisms on two levels. First, the data support that synaptogenic proteins guide connectivity and can function in distinct brain regions even if they are expressed broadly
Preparing CAM-SE for Multi-Tracer Applications: CAM-SE-Cslam
NASA Astrophysics Data System (ADS)
Lauritzen, P. H.; Taylor, M.; Goldhaber, S.
2014-12-01
The NCAR-DOE spectral element (SE) dynamical core comes from the HOMME (High-Order Modeling Environment; Dennis et al., 2012) and it is available in CAM. The CAM-SE dynamical core is designed with intrinsic mimetic properties guaranteeing total energy conservation (to time-truncation errors) and mass-conservation, and has demonstrated excellent scalability on massively parallel compute platforms (Taylor, 2011). For applications involving many tracers such as chemistry and biochemistry modeling, CAM-SE has been found to be significantly more computationally costly than the current "workhorse" model CAM-FV (Finite-Volume; Lin 2004). Hence a multi-tracer efficient scheme, called the CSLAM (Conservative Semi-Lagrangian Multi-tracer; Lauritzen et al., 2011) scheme, has been implemented in the HOMME (Erath et al., 2012). The CSLAM scheme has recently been cast in flux-form in HOMME so that it can be coupled to the SE dynamical core through conventional flux-coupling methods where the SE dynamical core provides background air mass fluxes to CSLAM. Since the CSLAM scheme makes use of a finite-volume gnomonic cubed-sphere grid and hence does not operate on the SE quadrature grid, the capability of running tracer advection, the physical parameterization suite and dynamics on separate grids has been implemented in CAM-SE. The default CAM-SE-CSLAM setup is to run physics on the quasi-equal area CSLAM grid. The capability of running physics on a different grid than the SE dynamical core may provide a more consistent coupling since the physics grid option operates with quasi-equal-area cell average values rather than non-equi-distant grid-point (SE quadrature point) values. Preliminary results on the performance of CAM-SE-CSLAM will be presented.
NASA Astrophysics Data System (ADS)
Deleuze, M. D.; Bernardi, P. B.; Caïs, Ph. C.; Perez, R. P.; Rees, J. M. R.; Pares, L. P.; Dubois, B. D.; Parot, Y. P.; Quertier, B. Q.; Maurice, S. M.; Maccabe, K. M.; Wiens, R. W.; Rull, F. R.
2016-10-01
This paper will describe and give a development status of SuperCam's mast unit. SuperCam will be carried on the Mars 2020 rover, and consists in an enhanced version of the ChemCam LIBS which is still performing at the surface of Mars, on Curiosity.
Glatthorn, Raymond H.
1986-01-01
A cam-controlled boring bar system (100) includes a first housing (152) which is rotatable about its longitudinal axis (154), and a second housing in the form of a cam-controlled slide (158) which is also rotatable about the axis (154) as well as being translatable therealong. A tool-holder (180) is mounted within the slide (158) for holding a single point cutting tool. Slide (158) has a rectangular configuration and is disposed within a rectangularly configured portion of the first housing (152). Arcuate cam slots (192) are defined within a side plate (172) of the housing (152), while cam followers (194) are mounted upon the cam slide (158) for cooperative engagement with the cam slots (192). In this manner, as the housing (152) and slide (158) rotate, and as the slide (158) also translates, a through-bore (14) having an hourglass configuration will be formed within a workpiece (16) which may be, for example, a nuclear reactor steam generator tube support plate.
Detection of Z DNA binding proteins in tissue culture cells.
Leith, I R; Hay, R T; Russell, W C
1988-01-01
A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA. Images PMID:3419919
Comparative genomics and evolution of the amylase-binding proteins of oral streptococci.
Haase, Elaine M; Kou, Yurong; Sabharwal, Amarpreet; Liao, Yu-Chieh; Lan, Tianying; Lindqvist, Charlotte; Scannapieco, Frank A
2017-04-20
Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.
Godsil, E.C.; Robinson, E.Y.
1963-07-16
A variable-throw cam comprising inner and outer eccentric sleeves which are adjustably locked together is described. The cam throw is varied by unlocking the inner and outer sleeves, rotating the outer sleeve relative to the inner one until the desired throw is obtained, and locking the sleeves together again. The cam is useful in applications wherein a continuously-variable throw is required, e.g., ram-and-die pressing operations, cyclic fatigue testing of materials, etc. (AEC)
Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin
2015-11-01
The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.
Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael
2011-01-01
Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for
Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J
2000-08-01
The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.
Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen
2015-05-29
Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.
SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.
Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa
2016-04-07
The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.
Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides
Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.
2016-01-01
The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114
Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding
DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.
Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less
Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding
Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.; ...
2017-09-06
Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less
Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor
2000-01-01
Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889
A Plasmodium falciparum copper-binding membrane protein with copper transport motifs
2012-01-01
Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769
de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M
2013-04-07
We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.
Cellulose binding domain fusion proteins
Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.
1998-02-17
A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.
Cellulose binding domain fusion proteins
Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.
1998-01-01
A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.
Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.
Schneider, G J; Geiduschek, E P
1990-06-25
The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.
Yamashita, Masashi; Sueyoshi, Noriyuki; Yamada, Hiroki; Katayama, Syouichi; Senga, Yukako; Takenaka, Yasuhiro; Ishida, Atsuhiko; Kameshita, Isamu; Shigeri, Yasushi
2018-04-20
We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca 2+ /CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca 2+ /CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca 2+ -signaling in C. cinerea.
Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer
van der Gun, B T F; Huisman, C; Stolzenburg, S; Kazemier, H G; Ruiters, M H J; Blancafort, P; Rots, M G
2013-01-01
Background: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear. Methods: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain. Results: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival. Conclusion: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer. PMID:23403823
Torabi, F; Bogle, O A; Estanyol, J M; Oliva, R; Miller, D
2017-12-01
Are there novel hyaladherins in human sperm? Zona pellucida-binding protein 2 (ZPBP2), containing a Link-like hyaluronic acid (HA)-binding domain, and several other proteins containing BX7B motifs, such as ADAM32 and Midkine, may be novel hyaladherins with HA-binding properties. HA-binding proteins (hyaladherins), which can bind HA surrounding the cumulus-oophorus complex, are distinct from hyases such as PH 20 (SPAM1) and are expressed by mature spermatozoa. Although HABP1 and CD44 are reasonably well characterized hyaladherins and the former has been implicated in sperm-oocyte interactions, the overall significance of sperm hyaladherins for male fertility is still poorly understood. This was a laboratory-based investigation into human sperm hyaladherins undertaken as part of a three year PhD programme sponsored by the EU Marie Curie Training network, Reprotrain. Protein homogenates of sperm obtained from young men of unknown fertility (N = 4) were partitioned into HA-binding and non-binding fractions by a protein affinity 'panning' method; their subsequent characterization was by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and partitioning behaviour was confirmed by western blotting. Sequences of proteins from both fractions were submitted to PDBsum to look for orthologous entries (PDB codes) and all returned codes were queried against the matching protein using SAS (Sequences Annotated by Structure) looking for structural similarities between them. A systematic search for other common features of hyaladherins was also undertaken. The presence of BX7B sequence motifs found in several well-described hyaladherins including RHAMM was used to assess efficacy of potential hyaladherin partitioning by the HA substrate. The data showed that 50% (14/28) and 34.5% (28/81) of proteins in the bound and unbound fractions, respectively, contained these motifs (one-tailed Z-score = 1.45; P = 0.074), indicating weak discrimination by the substrate. Querying PDBsum
Shorofi, Seyed Afshin; Arbon, Paul
2017-05-01
This study was intended to examine CAM among Australian hospital-based nurses, identifying their knowledge, attitude, personal and professional use, reasons for use, CAM referrals, and socio-demographic predictors of CAM users. Nurses holding a qualification in nursing and working in surgical wards were included using a convenience sampling technique. A self-complete questionnaire was developed to achieve the aims of the study. Descriptive and non-parametric statistics were calculated to describe and analyse data. Overall, 95.7% and 49.7% of nurses reported personal and professional use of CAM, respectively. The most popular CAM/CAM domain personally and professionally used by nurses was massage therapy and mind-body therapies. The primary reason for personal use of CAM was "[it] fits into my way of life/philosophy". Furthermore, massage therapists were the most commonly recommended CAM practitioners to patients. Only 15.8% of nurses would always ask patients about use of herbal medicines as part of nursing history taking. Over one-fifth (22.4%) of nurses rated their attitude as having a very positive, and 60.3% rated themselves as having very little or no knowledge of CAM. A positive correlation was also found between knowledge and attitude about CAM. Positive attitude and higher knowledge about CAM were positively correlated to CAM referrals. Several socio-demographic factors predicted personal and professional use of CAM. This study revealed that nurses generally believe not to have sufficient knowledge of CAM but are open to use CAM with patients. Nurses' positive attitude toward and personal use of CAM could be an indication that they are poised for further integration of evidence-based CAM into nursing practice to treat whole person. Copyright © 2017 Elsevier Ltd. All rights reserved.
Deciphering Cryptic Binding Sites on Proteins by Mixed-Solvent Molecular Dynamics.
Kimura, S Roy; Hu, Hai Peng; Ruvinsky, Anatoly M; Sherman, Woody; Favia, Angelo D
2017-06-26
In recent years, molecular dynamics simulations of proteins in explicit mixed solvents have been applied to various problems in protein biophysics and drug discovery, including protein folding, protein surface characterization, fragment screening, allostery, and druggability assessment. In this study, we perform a systematic study on how mixtures of organic solvent probes in water can reveal cryptic ligand binding pockets that are not evident in crystal structures of apo proteins. We examine a diverse set of eight PDB proteins that show pocket opening induced by ligand binding and investigate whether solvent MD simulations on the apo structures can induce the binding site observed in the holo structures. The cosolvent simulations were found to induce conformational changes on the protein surface, which were characterized and compared with the holo structures. Analyses of the biological systems, choice of probes and concentrations, druggability of the resulting induced pockets, and application to drug discovery are discussed here.
The binding of sodium dodecyl sulphate to various proteins
Pitt-Rivers, Rosalind; Impiombato, F. S. Ambesi
1968-01-01
1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90–100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70–100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S·S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S·S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein–sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000–41000) irrespective of the molecular weight of the protein to which they were attached. PMID:4177067
Cornell, Thomas A; Fu, Jing; Newland, Stephanie H; Orner, Brendan P
2013-11-06
Proteins that form cage-like structures have been of much recent cross-disciplinary interest due to their application to bioconjugate and materials chemistry, their biological functions spanning multiple essential cellular processes, and their complex structure, often defined by highly symmetric protein–protein interactions. Thus, establishing the fundamentals of their formation, through detecting and quantifying important protein–protein interactions, could be crucial to understanding essential cellular machinery, and for further development of protein-based technologies. Herein we describe a method to monitor the assembly of protein cages by detecting specific, oligomerization state dependent, protein–protein interactions. Our strategy relies on engineering protein monomers to include cysteine pairs that are presented proximally if the cage state assembles. These assembled pairs of cysteines act as binding sites for the fluorescent reagent FlAsH, which, once bound, provides a readout for successful oligomerization. As a proof of principle, we applied this technique to the iron storage protein, DNA-binding protein from starved cells from E. coli. Several linker lengths and conformations for the presentation of the cysteine pairs were screened to optimize the engineered binding sites. We confirmed that our designs were successful in both lysates and with purified proteins, and that FlAsH binding was dependent upon cage assembly. Following successful characterization of the assay, its throughput was expanded. A two-dimension matrix of pH and denaturing buffer conditions was screened to optimize nanocage stability. We intend to use this method for the high throughput screening of protein cage libraries and of conditions for the generation of inorganic nanoparticles within the cavity of these and other cage proteins.
Profiling Charge Complementarity and Selectivity for Binding at the Protein Surface
Sulea, Traian; Purisima, Enrico O.
2003-01-01
A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins. PMID:12719221
Profiling charge complementarity and selectivity for binding at the protein surface.
Sulea, Traian; Purisima, Enrico O
2003-05-01
A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.
Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth Manduca sexta.
Lohr, Christian; Bergstein, Sandra; Hirnet, Daniela
2007-01-01
The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6-10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.
Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling
DOE Office of Scientific and Technical Information (OSTI.GOV)
Winder, B.S.
1988-01-01
Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) formore » binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.« less
Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.
Dyson, M H; Rose, S; Mahadevan, L C
2001-08-01
Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.
Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K
2009-05-01
Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.
Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.
Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K
2006-11-01
We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.
A major integral protein of the plant plasma membrane binds flavin.
Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer
2003-05-01
Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.
Medrano, Francisco Javier; de Souza, Cristiane Santos; Romero, Antonio; Balan, Andrea
2014-01-01
The uptake of maltose and related sugars in Gram-negative bacteria is mediated by an ABC transporter encompassing a periplasmic component (the maltose-binding protein or MalE), a pore-forming membrane protein (MalF and MalG) and a membrane-associated ATPase (MalK). In the present study, the structure determination of the apo form of the putative maltose/trehalose-binding protein (Xac-MalE) from the citrus pathogen Xanthomonas citri in space group P6522 is described. The crystals contained two protein molecules in the asymmetric unit and diffracted to 2.8 Å resolution. Xac-MalE conserves the structural and functional features of sugar-binding proteins and a ligand-binding pocket with similar characteristics to eight different orthologues, including the residues for maltose and trehalose interaction. This is the first structure of a sugar-binding protein from a phytopathogenic bacterium, which is highly conserved in all species from the Xanthomonas genus. PMID:24817711
Characterization of auxin-binding proteins from zucchini plasma membrane
NASA Technical Reports Server (NTRS)
Hicks, G. R.; Rice, M. S.; Lomax, T. L.
1993-01-01
We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may
Characterization of auxin-binding proteins from zucchini plasma membrane.
Hicks, G R; Rice, M S; Lomax, T L
1993-01-01
We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may
Collective Dynamics of Periplasmic Glutamine Binding Protein upon Domain Closure
Loeffler, Hannes H.; Kitao, Akio
2009-01-01
The glutamine binding protein is a vital component of the associated ATP binding cassette transport systems responsible for the uptake of glutamine into the cell. We have investigated the global movements of this protein by molecular dynamics simulations and principal component analysis (PCA). We confirm that the most dominant mode corresponds to the biological function of the protein, i.e., a hinge-type motion upon ligand binding. The closure itself was directly observed from two independent trajectories whereby PCA was used to elucidate the nature of this closing reaction. Two intermediary states are identified and described in detail. The ligand binding induces the structural change of the hinge regions from a discontinuous β-sheet to a continuous one, which also enhances softness of the hinge and modifies the direction of hinge motion to enable closing. We also investigated the convergence behavior of PCA modes, which were found to converge rather quickly when the associated magnitudes of the eigenvalues are well separated. PMID:19883597
Nichol, James; Thompson, Elizabeth A; Shaw, Alison
2011-02-01
The rise in complementary and alternative medicine (CAM) use is well documented. Surveys provide varying estimates of the prevalence of CAM use. Qualitative research has explored individuals' decision-making regarding CAM. This study aimed to examine the family as a context for beliefs, decision-making, and dialogue about CAM. Families were recruited via the Avon Longitudinal Study of Parents and Children. A subsample of CAM users was targeted using purposeful sampling. Focus groups and interviews were conducted with 15 families and the data were analyzed thematically. Family understandings and beliefs about CAM: CAM was understood as treatments provided outside mainstream care, offering a more "natural" and "holistic" approach, tailored to individual needs and overlapping with wider healthy lifestyle practices. Hierarchies of acceptability of CAM: Physical and "mainstream" therapies were widely supported, with "fringe" therapies producing the most polarized views. There was a belief particularly among fathers and young people that certain therapies rely on "placebo" effects and their value was contested. Types of CAM users within families: Family members were predominantly "pragmatic" CAM users, with "committed" users (all mothers) characterized by deeper philosophical commitment to CAM and skepticism toward conventional medicine. Family dynamics of CAM decision-making: Mothers tended to "champion" CAM within families, while not determining family CAM use. Fathers largely positioned themselves as lacking expertise or skeptical of CAM. Young people were beginning to articulate independent and more critical views of CAM, some directly challenging their mother's perspective. However, all families shared openness to CAM as part of broader beliefs in proactive healthy lifestyles. Family focus groups and interviews allow a window on beliefs, decision-making, and dialogue about CAM within families, illuminating the CAM "champion" role held by mothers, and young people
Chui, Ping Lei; Abdullah, Khatijah Lim; Wong, Li Ping; Taib, Nur Aishah
2015-01-01
Background Complementary and alternative medicine (CAM) use has become increasingly popular among patients with cancer. The purposes of this study were to compare the QOL in CAM users and non-CAM users and to determine whether CAM use influences QOL among breast cancer patients during chemotherapy. Methodology A cross-sectional survey was conducted at two outpatient chemotherapy centers. A total of 546 patients completed the questionnaires on CAM use. QOL was evaluated based on the European Organization for Research and Treatment of Cancer (EORTC) core quality of life (QLQ-C30) and breast cancer-specific quality of life (QLQ-BR23) questionnaires. Results A total of 70.7% of patients were identified as CAM users. There was no significant difference in global health status scores and in all five subscales of the QLQ C30 functional scales between CAM users and non-CAM users. On the QLQ-C30 symptom scales, CAM users (44.96±3.89) had significantly (p = 0.01) higher mean scores for financial difficulties than non-CAM users (36.29±4.81). On the QLQ-BR23 functional scales, CAM users reported significantly higher mean scores for sexual enjoyment (6.01±12.84 vs. 4.64±12.76, p = 0.04) than non-CAM users. On the QLQ-BR23 symptom scales, CAM users reported higher systemic therapy side effects (41.34±2.01 vs. 37.22±2.48, p = 0.04) and breast symptoms (15.76±2.13 vs. 11.08±2.62, p = 0.02) than non-CAM users. Multivariate logistic regression analysis indicated that the use of CAM modality was not significantly associated with higher global health status scores (p = 0.71). Conclusion While the findings indicated that there was no significant difference between users and non-users of CAM in terms of QOL, CAM may be used by health professionals as a surrogate to monitor patients with higher systemic therapy side effects and breast symptoms. Furthermore, given that CAM users reported higher financial burdens (which may have contributed to increased distress), patients should
DOE Office of Scientific and Technical Information (OSTI.GOV)
Giulliani, S. E.; Frank, A. E.; Collart, F. R.
2008-12-08
We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less
CLASPs are required for proper microtubule localization of End-binding proteins
Grimaldi, Ashley D.; Maki, Takahisa; Fitton, Benjamin P.; Roth, Daniel; Yampolsky, Dmitry; Davidson, Michael W.; Svitkina, Tatyana; Straube, Anne; Hayashi, Ikuko; Kaverina, Irina
2014-01-01
Summary Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus-ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that Cytoplasmic linker associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus-ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, while neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at pre-formed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP-hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells. PMID:25117684
Calculation of protein-ligand binding affinities.
Gilson, Michael K; Zhou, Huan-Xiang
2007-01-01
Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.
Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.
Sass-Kuhn, S P; Moqbel, R; Mackay, J A; Cromwell, O; Kay, A B
1984-01-01
Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter. Images PMID:6690479
Ogawara, Hiroshi
2016-09-01
PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.
Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi
2016-11-01
To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.
Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke
2014-01-01
Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID
The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation.
Catalá, A
1984-10-01
Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.
Identification of DNA-binding proteins using structural, electrostatic and evolutionary features.
Nimrod, Guy; Szilágyi, András; Leslie, Christina; Ben-Tal, Nir
2009-04-10
DNA-binding proteins (DBPs) participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance. We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions (patches) on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein, including its dipole moment. Using 10-fold cross-validation on a dataset of 138 DBPs and 110 proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0.90, which is overall better than the performance of published methods. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly. The resulting classifier was applied to a collection of 757 proteins of known structure and unknown function. Of these proteins, 218 were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.
A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.
Banasik, Michał; Sachadyn, Paweł
2016-09-01
A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.
Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne
2011-12-01
During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.
Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael
2011-01-01
Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful
Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302).
Watanabe, Tanya A; Geary, Richard S; Levin, Arthur A
2006-01-01
In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.
Gavert, Nancy; Ben-Shmuel, Amir; Raveh, Shani; Ben-Ze'ev, Avri
2008-11-01
L1-cell adhesion molecule (L1-CAM) is a cell adhesion receptor of the immunoglobulin superfamily, known for its roles in nerve cell function. While originally believed to be present only in brain cells, in recent years L1-CAM has been detected in other tissues, and in a variety of cancer cells, including some common types of human cancer. We review the prevalence of L1-CAM in human cancer, the possible mechanisms involved in L1-CAM-mediated tumorigenesis, and cancer therapies based upon L1-CAM antibody treatment. In colon cancer cells, the L1-CAM gene was identified as a target of the Wnt/beta-catenin-TCF signaling pathway, and L1-CAM was exclusively detected at the invasive front of colon and ovarian cancer tissue. The expression of L1-CAM in normal and cancer cells enhanced tumorigenesis and conferred metastasis in colon cancer cells. Antibodies against the L1-CAM ectodomain severely inhibited the proliferation of a variety of cancer cells in culture and reduced tumor burden when injected into mice harboring cancer cells expressing L1-CAM. These results, in addition to the presence of L1-CAM on the cell surface and its restricted distribution in normal tissues, make it an ideal target for tumor therapy.
Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S
1988-01-01
The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996
A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.
Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua
2014-12-01
We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.
Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M
2015-10-01
Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.
Gold, Nicola D; Jackson, Richard M
2006-02-03
The rapid growth in protein structural data and the emergence of structural genomics projects have increased the need for automatic structure analysis and tools for function prediction. Small molecule recognition is critical to the function of many proteins; therefore, determination of ligand binding site similarity is important for understanding ligand interactions and may allow their functional classification. Here, we present a binding sites database (SitesBase) that given a known protein-ligand binding site allows rapid retrieval of other binding sites with similar structure independent of overall sequence or fold similarity. However, each match is also annotated with sequence similarity and fold information to aid interpretation of structure and functional similarity. Similarity in ligand binding sites can indicate common binding modes and recognition of similar molecules, allowing potential inference of function for an uncharacterised protein or providing additional evidence of common function where sequence or fold similarity is already known. Alternatively, the resource can provide valuable information for detailed studies of molecular recognition including structure-based ligand design and in understanding ligand cross-reactivity. Here, we show examples of atomic similarity between superfamily or more distant fold relatives as well as between seemingly unrelated proteins. Assignment of unclassified proteins to structural superfamiles is also undertaken and in most cases substantiates assignments made using sequence similarity. Correct assignment is also possible where sequence similarity fails to find significant matches, illustrating the potential use of binding site comparisons for newly determined proteins.
The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.
Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan
2014-10-13
The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.
Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie
2015-06-21
Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.
Membrane proteins bind lipids selectively to modulate their structure and function.
Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V
2014-06-05
Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane
RNA-Binding Proteins Revisited - The Emerging Arabidopsis mRNA Interactome.
Köster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee
2017-06-01
RNA-protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture - where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes. Copyright © 2017 Elsevier Ltd. All rights reserved.
PI(4,5)P2-binding effector proteins for vesicle exocytosis
Martin, Thomas F. J.
2014-01-01
PI(4,5)P2 participates directly in priming and possibly fusion steps of Ca2+-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2 reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2 directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2 effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2 in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. PMID:25280637
Dual Role of Protein Phosphorylation in DNA Activator/Coactivator Binding
Dadarlat, Voichita M.; Skeel, Robert D.
2011-01-01
Binding free energies are calculated for the phosphorylated and unphosphorylated complexes between the kinase inducible domain (KID) of the DNA transcriptional activator cAMP response element binding (CREB) protein and the KIX domain of its coactivator, CREB-binding protein (CBP). To our knowledge, this is the first application of a method based on a potential of mean force (PMF) with restraining potentials to compute the binding free energy of protein-protein complexes. The KID:KIX complexes are chosen here because of their biological relevance to the DNA transcription process and their relatively small size (81 residues for the KIX domain of CBP, and 28 residues for KID). The results for pKID:KIX and KID:KIX are −9.55 and −4.96 kcal/mol, respectively, in good agreement with experimental estimates (−8.8 and −5.8 kcal/mol, respectively). A comparison between specific contributions to protein-protein binding for the phosphorylated and unphosphorylated complexes reveals a dual role for the phosphorylation of KID at Ser-133 in effecting a more favorable free energy of the bound system: 1), stabilization of the unbound conformation of phosphorylated KID due to favorable intramolecular interactions of the phosphate group of Ser-133 with the charged groups of an arginine-rich region spanning both α-helices, which lowers the configurational entropy; and 2), more favorable intermolecular electrostatic interactions between pSer-133 and Arg-131 of KID, and Lys-662, Tyr-658, and Glu-666 of KIX. Charge reduction through ligand phosphorylation emerges as a possible mechanism for controlling the unbound state conformation of KID and, ultimately, gene expression. This work also demonstrates that the PMF-based method with restraining potentials provides an added benefit in that important elements of the binding pathway are evidenced. Furthermore, the practicality of the PMF-based method for larger systems is validated by agreement with experiment. In addition, we provide
Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding
Sirois, Allison R.; Deny, Daniela A.; Baierl, Samantha R.; George, Katia S.
2018-01-01
Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics. PMID:29738555
Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y
1997-02-01
Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.
Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M
1992-02-01
The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs
Yugandhar, K; Gromiha, M Michael
2014-09-01
Protein-protein interactions are intrinsic to virtually every cellular process. Predicting the binding affinity of protein-protein complexes is one of the challenging problems in computational and molecular biology. In this work, we related sequence features of protein-protein complexes with their binding affinities using machine learning approaches. We set up a database of 185 protein-protein complexes for which the interacting pairs are heterodimers and their experimental binding affinities are available. On the other hand, we have developed a set of 610 features from the sequences of protein complexes and utilized Ranker search method, which is the combination of Attribute evaluator and Ranker method for selecting specific features. We have analyzed several machine learning algorithms to discriminate protein-protein complexes into high and low affinity groups based on their Kd values. Our results showed a 10-fold cross-validation accuracy of 76.1% with the combination of nine features using support vector machines. Further, we observed accuracy of 83.3% on an independent test set of 30 complexes. We suggest that our method would serve as an effective tool for identifying the interacting partners in protein-protein interaction networks and human-pathogen interactions based on the strength of interactions. © 2014 Wiley Periodicals, Inc.
Conacci-Sorrell, Maralice; Kaplan, Anna; Raveh, Shani; Gavert, Nancy; Sakurai, Takeshi; Ben-Ze'ev, Avri
2005-12-15
Nr-CAM, a cell-cell adhesion molecule of the immunoglobulin-like cell adhesion molecule family, known for its function in neuronal outgrowth and guidance, was recently identified as a target gene of beta-catenin signaling in human melanoma and colon carcinoma cells and tissue. Retrovirally mediated transduction of Nr-CAM into fibroblasts induces cell motility and tumorigenesis. We investigated the mechanisms by which Nr-CAM can confer properties related to tumor cell behavior and found that Nr-CAM expression in NIH3T3 cells protects cells from apoptosis in the absence of serum by constitutively activating the extracellular signal-regulated kinase and AKT signaling pathways. We detected a metalloprotease-mediated shedding of Nr-CAM into the culture medium of cells transfected with Nr-CAM, and of endogenous Nr-CAM in B16 melanoma cells. Conditioned medium and purified Nr-CAM-Fc fusion protein both enhanced cell motility, proliferation, and extracellular signal-regulated kinase and AKT activation. Moreover, Nr-CAM was found in complex with alpha4beta1 integrins in melanoma cells, indicating that it can mediate, in addition to homophilic cell-cell adhesion, heterophilic adhesion with extracellular matrix receptors. Suppression of Nr-CAM levels by small interfering RNA in B16 melanoma inhibited the adhesive and tumorigenic capacities of these cells. Stable expression of the Nr-CAM ectodomain in NIH3T3 cells conferred cell transformation and tumorigenesis in mice, suggesting that the metalloprotease-mediated shedding of Nr-CAM is a principal route for promoting oncogenesis by Nr-CAM.
Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H
2016-09-06
Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.
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
Astegno, Alessandra; La Verde, Valentina; Marino, Valerio; Dell'Orco, Daniele; Dominici, Paola
2016-03-01
In plants, transient elevation of intracellular Ca(2+) concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~20 μM apparent affinity for Ca(2+) and the C-lobe shows a ~6-fold higher affinity than N-lobe (Kd(app) 5.6 μM and 32 μM for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca(2+) in a manner similar to vertebrate CaM based on comparison of Ca(2+)-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca(2+)-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holoprotein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD. Copyright © 2015 Elsevier B.V. All rights reserved.
Role of Electrostatics in Protein-RNA Binding: The Global vs the Local Energy Landscape.
Ghaemi, Zhaleh; Guzman, Irisbel; Gnutt, David; Luthey-Schulten, Zaida; Gruebele, Martin
2017-09-14
U1A protein-stem loop 2 RNA association is a basic step in the assembly of the spliceosomal U1 small nuclear ribonucleoprotein. Long-range electrostatic interactions due to the positive charge of U1A are thought to provide high binding affinity for the negatively charged RNA. Short range interactions, such as hydrogen bonds and contacts between RNA bases and protein side chains, favor a specific binding site. Here, we propose that electrostatic interactions are as important as local contacts in biasing the protein-RNA energy landscape toward a specific binding site. We show by using molecular dynamics simulations that deletion of two long-range electrostatic interactions (K22Q and K50Q) leads to mutant-specific alternative RNA bound states. One of these states preserves short-range interactions with aromatic residues in the original binding site, while the other one does not. We test the computational prediction with experimental temperature-jump kinetics using a tryptophan probe in the U1A-RNA binding site. The two mutants show the distinct predicted kinetic behaviors. Thus, the stem loop 2 RNA has multiple binding sites on a rough RNA-protein binding landscape. We speculate that the rough protein-RNA binding landscape, when biased to different local minima by electrostatics, could be one way that protein-RNA interactions evolve toward new binding sites and novel function.
Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes
Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.
2005-01-01
Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465
Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.
2016-01-01
Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287
NASA Technical Reports Server (NTRS)
Carter, Daniel C. (Inventor)
1998-01-01
In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.
FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.
Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane
2007-03-01
Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.
Sampling protein motion and solvent effect during ligand binding
Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele
2012-01-01
An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423
Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J
2008-10-01
Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.
Sampling and energy evaluation challenges in ligand binding protein design.
Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David
2017-12-01
The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.
Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins
Kfoury, N.; Kapatos, G.
2009-01-01
CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292
Cloud computing for protein-ligand binding site comparison.
Hung, Che-Lun; Hua, Guan-Jie
2013-01-01
The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.
Tsukamoto, Hisao; Farrens, David L.
2013-01-01
G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics. PMID:23940032
Tsukamoto, Hisao; Farrens, David L
2013-09-27
G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.
Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast*
Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun
2012-01-01
Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017
Efficient identification of tubby-binding proteins by an improved system of T7 phage display.
Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei
2010-01-01
Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.
UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kataoka, H.; Fujiwara, Y.
1991-03-29
The gel mobility shift assay method revealed a specifically ultraviolet (UV) damage recognizing, DNA-binding protein in nuclear extracts of normal human cells. The resulted DNA/protein complexes caused the two retarded mobility shifts. Four xeroderma pigmentosum complementation group E (XPE) fibroblast strains derived from unrelated Japanese families were not deficient in such a DNA damage recognition/binding protein because of the normal complex formation and gel mobility shifts, although we confirmed the reported lack of the protein in the European XPE (XP2RO and XP3RO) cells. Thus, the absence of this binding protein is not always commonly observed in all the XPE strains,more » and the partially repair-deficient and intermediately UV-hypersensitive phenotype of XPE cells are much similar whether or not they lack the protein.« less
Carotenoid Antenna Binding and Function in Retinal Proteins
2012-08-13
REPORT Carotenoid antenna binding and function in retinal proteins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Xanthorhodopsin, a proton pump from the...eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light... carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4
A web server for analysis, comparison and prediction of protein ligand binding sites.
Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S
2016-03-25
One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .
Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin
2017-08-01
Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.