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Sample records for acidic coiled-coil protein

  1. Engineered coiled-coil protein microfibers.

    PubMed

    Hume, Jasmin; Sun, Jennifer; Jacquet, Rudy; Renfrew, P Douglas; Martin, Jesse A; Bonneau, Richard; Gilchrist, M Lane; Montclare, Jin Kim

    2014-10-13

    The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.

  2. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    PubMed

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.

  3. A periodic table of coiled-coil protein structures.

    PubMed

    Moutevelis, Efrosini; Woolfson, Derek N

    2009-01-23

    Coiled coils are protein structure domains with two or more alpha-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a "periodic table of coiled coils," which we have made available at http://coiledcoils.chm.bris.ac.uk/ccplus/search/periodic_table. In this table, coiled-coil assemblies are arranged in columns with increasing numbers of alpha-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.

  4. Mitochondrial Proteins Containing Coiled-Coil-Helix-Coiled-Coil-Helix (CHCH) Domains in Health and Disease.

    PubMed

    Modjtahedi, Nazanine; Tokatlidis, Kostas; Dessen, Philippe; Kroemer, Guido

    2016-03-01

    Members of the coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing protein family that carry (CX9C) type motifs are imported into the mitochondrion with the help of the disulfide relay-dependent MIA import pathway. These evolutionarily conserved proteins are emerging as new cellular factors that control mitochondrial respiration, redox regulation, lipid homeostasis, and membrane ultrastructure and dynamics. We discuss recent insights on the activity of known (CX9C) motif-carrying proteins in mammals and review current data implicating the Mia40/CHCHD4 import machinery in the regulation of their mitochondrial import. Recent findings and the identification of disease-associated mutations in specific (CX9C) motif-carrying proteins have highlighted members of this family of proteins as potential therapeutic targets in a variety of human disorders.

  5. Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe.

    PubMed

    Ohtaka, Ayami; Saito, Takamune T; Okuzaki, Daisuke; Nojima, Hiroshi

    2007-05-18

    Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which play essential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 and Mcp7 are required for their function as a putative recombinase cofactor complex during meiotic recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required for horsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these proteins plays an important role during the progression of cells through meiosis. However, there are still many unanswered questions on how these proteins operate. In this paper, we briefly review recent studies on the meiotic coiled-coil proteins in Sz. pombe.

  6. Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe

    PubMed Central

    Ohtaka, Ayami; Saito, Takamune T; Okuzaki, Daisuke; Nojima, Hiroshi

    2007-01-01

    Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which play essential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 and Mcp7 are required for their function as a putative recombinase cofactor complex during meiotic recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required for horsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these proteins plays an important role during the progression of cells through meiosis. However, there are still many unanswered questions on how these proteins operate. In this paper, we briefly review recent studies on the meiotic coiled-coil proteins in Sz. pombe. PMID:17509158

  7. Transport Vesicle Tethering at the Trans Golgi Network: Coiled Coil Proteins in Action

    PubMed Central

    Cheung, Pak-yan P.; Pfeffer, Suzanne R.

    2016-01-01

    The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed. PMID:27014693

  8. Repeats in transforming acidic coiled-coil (TACC) genes.

    PubMed

    Trivedi, Seema

    2013-06-01

    Transforming acidic coiled-coil proteins (TACC1, 2, and 3) are essential proteins associated with the assembly of spindle microtubules and maintenance of bipolarity. Dysregulation of TACCs is associated with tumorigenesis, but studies of microsatellite instability in TACC genes have not been extensive. Microsatellite or simple sequence repeat instability is known to cause many types of cancer. The present in silico analysis of SSRs in human TACC gene sequences shows the presence of mono- to hexa-nucleotide repeats, with the highest densities found for mono- and di-nucleotide repeats. Density of repeats is higher in introns than in exons. Some of the repeats are present in regulatory regions and retained introns. Human TACC genes show conservation of many repeat classes. Microsatellites in TACC genes could be valuable markers for monitoring numerical chromosomal aberrations and or cancer.

  9. Functional investigation of the plant-specific long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana.

    PubMed

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response.

  10. Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

    PubMed Central

    Venkatakrishnan, Sowmya; Mackey, David; Meier, Iris

    2013-01-01

    We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response. PMID:23451199

  11. Structural and biochemical characterizations of an intramolecular tandem coiled coil protein.

    PubMed

    Shin, Donghyuk; Kim, Gwanho; Kim, Gyuhee; Zheng, Xu; Kim, Yang-Gyun; Lee, Sangho

    2014-12-12

    Coiled coil has served as an excellent model system for studying protein folding and developing protein-based biomaterials. Most designed coiled coils function as oligomers, namely intermolecular coiled coils. However, less is known about structural and biochemical behavior of intramolecular coiled coils where coiled coil domains are covalently linked in one polypeptide. Here we prepare a protein which harbors three coiled coil domains with two short linkers, termed intramolecular tandem coiled coil (ITCC) and characterize its structural and biochemical behavior in solution. ITCC consists of three coiled coil domains whose sequences are derived from Coil-Ser and its domain swapped dimer. Modifications include positioning E (Glu) residue at "e" and K (Lys) at "g" positions throughout heptad repeats to enhance ionic interaction among its constituent coiled coil domains. Molecular modeling of ITCC suggests a compact triple helical bundle structure with the second and the third coiled coil domains forming a canonical coiled coil. ITCC exists as a mixture of monomeric and dimeric species in solution. Small-angle X-ray scattering reveals ellipsoidal molecular envelopes for both dimeric and monomeric ITCC in solution. The theoretically modeled structures of ITCC dock well into the envelopes of both species. Higher ionic strength shifts the equilibrium into monomer with apparently more compact structure while secondary structure remains unchanged. Taken together, our results suggest that our designed ITCC is predominantly monomeric structure through the enhanced ionic interactions, and its conformation is affected by the concentration of ionic species in the buffer.

  12. Solving coiled-coil protein structures

    SciTech Connect

    Dauter, Zbigniew

    2015-02-26

    With the availability of more than 100,000 entries stored in the Protein Data Bank (PDB) that can be used as search models, molecular replacement (MR) is currently the most popular method of solving crystal structures of macromolecules. Significant methodological efforts have been directed in recent years towards making this approach more powerful and practical. This resulted in the creation of several computer programs, highly automated and user friendly, that are able to successfully solve many structures even by researchers who, although interested in structures of biomolecules, are not very experienced in crystallography.

  13. A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.

    PubMed

    Simpson, Raina J Y; Yi Lee, Stella Hoi; Bartle, Natalie; Sum, Eleanor Y; Visvader, Jane E; Matthews, Jacqueline M; Mackay, Joel P; Crossley, Merlin

    2004-09-17

    Classic zinc finger domains (cZFs) consist of a beta-hairpin followed by an alpha-helix. They are among the most abundant of all protein domains and are often found in tandem arrays in DNA-binding proteins, with each finger contributing an alpha-helix to effect sequence-specific DNA recognition. Lone cZFs, not found in tandem arrays, have been postulated to function in protein interactions. We have studied the transcriptional co-regulator Friend of GATA (FOG), which contains nine zinc fingers. We have discovered that the third cZF of FOG contacts a coiled-coil domain in the centrosomal protein transforming acidic coiled-coil 3 (TACC3). Although FOG-ZF3 exhibited low solubility, we have used a combination of mutational mapping and protein engineering to generate a derivative that was suitable for in vitro and structural analysis. We report that the alpha-helix of FOG-ZF3 recognizes a C-terminal portion of the TACC3 coiled-coil. Remarkably, the alpha-helical surface utilized by FOG-ZF3 is the same surface responsible for the well established sequence-specific DNA-binding properties of many other cZFs. Our data demonstrate the versatility of cZFs and have implications for the analysis of many as yet uncharacterized cZF proteins.

  14. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    NASA Astrophysics Data System (ADS)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  15. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    SciTech Connect

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-10-31

    The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  16. Force modulated conductance of artificial coiled-coil protein monolayers.

    PubMed

    Atanassov, Alexander; Hendler, Ziv; Berkovich, Inbal; Ashkenasy, Gonen; Ashkenasy, Nurit

    2013-01-01

    Studies of charge transport through proteins bridged between two electrodes have been the subject of intense research in recent years. However, the complex structure of proteins makes it difficult to elucidate transport mechanisms, and the use of simple peptide oligomers may be an over simplified model of the proteins. To bridge this structural gap, we present here studies of charge transport through artificial parallel coiled-coil proteins conducted in dry environment. Protein monolayers uniaxially oriented at an angle of ∼ 30° with respect to the surface normal were prepared. Current voltage measurements, obtained using conductive-probe atomic force microscopy, revealed the mechano-electronic behavior of the protein films. It was found that the low voltage conductance of the protein monolayer increases linearly with applied force, mainly due to increase in the tip contact area. Negligible compression of the films for loads below 26 nN allowed estimating a tunneling attenuation factor, β(0) , of 0.5-0.6 Å(-1) , which is akin to charge transfer by tunneling mechanism, despite the comparably large charge transport distance. These studies show that mechano-electronic behavior of proteins can shed light on their complex charge transport mechanisms, and on how these mechanisms depend on the detailed structure of the proteins. Such studies may provide insightful information on charge transfer in biological systems.

  17. Pharmacological interference with protein-protein interactions mediated by coiled-coil motifs.

    PubMed

    Strauss, H M; Keller, S

    2008-01-01

    Coiled coils are bundles of intertwined alpha-helices that provide protein-protein interaction sites for the dynamic assembly and disassembly of protein complexes. The coiled-coil motif combines structural versatility and adaptability with mechanical strength and specificity. Multimeric proteins that rely on coiled-coil interactions are structurally and functionally very diverse, ranging from simple homodimeric transcription factors to elaborate heteromultimeric scaffolding clusters. Several coiled-coil-bearing proteins are of outstanding pharmacological importance, most notably SNARE proteins involved in vesicular trafficking of neurotransmitters and viral fusion proteins. Together with their crucial roles in many physiological and pathological processes, the structural simplicity and reversible nature of coiled-coil associations render them a promising target for pharmacological interference, as successfully exemplified by botulinum toxins and viral fusion inhibitors. The alpha-helical coiled coil is a ubiquitous protein domain that mediates highly specific homo- and heteromeric protein-protein interactions among a wide range of proteins. The coiled-coil motif was first proposed by Crick on the basis of X-ray diffraction data on alpha-keratin more than 50 years ago (Crick 1952, 1953) and nowadays belongs to the best-characterized protein interaction modules. By definition, a coiled coil is an oligomeric protein assembly consisting of several right-handed amphipathic alpha-helices that wind around each other into a superhelix (or a supercoil) in which the hydrophobic surfaces of the constituent helices are in continuous contact, forming a hydrophobic core. Both homomeric and heteromeric coiled coils with different stoichiometries are possible, and the helices can be aligned in either a parallel or an antiparallel topology (Harbury et al. 1993, 1994). Stoichiometry and topology are governed by the primary structure, that is, the sequence of the polypeptide chains

  18. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  19. The Golgin Family of Coiled-Coil Tethering Proteins

    PubMed Central

    Witkos, Tomasz M.; Lowe, Martin

    2016-01-01

    The golgins are a family of predominantly coiled-coil proteins that are localized to the Golgi apparatus. Golgins are present in all eukaryotes, suggesting an evolutionary conserved function. Golgins are anchored to the Golgi membrane by their carboxy terminus and are predicted to adopt an extended conformation that projects into the surrounding cytoplasm. This arrangement is ideal for the capture or tethering of nearby membranes or cytoskeletal elements. Golgin-mediated tethering is thought to be important for vesicular traffic at the Golgi apparatus, the maintenance of Golgi architecture, as well as the positioning of the Golgi apparatus within cells. In addition to acting as tethers, some golgins can also sequester various factors at the Golgi membrane, allowing for the spatiotemporal regulation of downstream cellular functions. Although it is now established that golgins are membrane and cytoskeleton tethers, the mechanisms underlying tethering remain poorly defined. Moreover, the importance of golgin-mediated tethering in a physiological context remains to be fully explored. This review will describe our current understanding of golgin function, highlighting recent progress that has been made, and goes on to discuss outstanding questions and potential avenues for future research with regard to this family of conserved Golgi-associated proteins. PMID:26793708

  20. Gas-phase IR spectra of intact [alpha]-helical coiled coil protein complexes

    NASA Astrophysics Data System (ADS)

    Pagel, Kevin; Kupser, Peter; Bierau, Frauke; Polfer, Nicolas C.; Steill, Jeffrey D.; Oomens, Jos; Meijer, Gerard; Koksch, Beate; von Helden, Gert

    2009-06-01

    Electrospray ionization (ESI) is the softest ionization method that is currently available and it is widely accepted, that ESI generated ions of proteins and protein assemblies at certain conditions retain characteristic aspects of their solution-state conformation. ESI mass spectrometry (MS) therefore evolved as a useful tool to obtain information on composition, stoichiometry, and dynamics of non-covalently associated protein complexes. While tertiary structure information of proteins can be obtained from ion mobility spectrometry (IMS), only a few techniques yield direct information on the secondary structure of gas-phase peptides and proteins. We present here the mid-IR spectroscopic secondary structural analysis of three de novo designed [alpha]-helical coiled coil model peptides and their non-covalently associated complexes in the gas-phase. The conformational stability of such coiled coil peptides in solution is primarily driven by aggregation. Isolated monomers usually remain unfolded. Two of the investigated peptides were designed to assemble into stable [alpha]-helical complexes in acidic solution, while the third one remains monomeric and unfolded at these conditions. Monomer ions of all three peptides show comparable photodissociation IR spectra and therefore suggest an unfolded conformation in the gas phase. In contrast, considerable CO stretch (amide-I) and N-H bend (amide-II) band shifts have been observed for the dimers which is consistent with an elevated H-bond content. These findings provide evidence that at least a fraction of the condensed phase [alpha]-helical structure is retained in the gas-phase coiled coil complexes.

  1. Critical evaluation of in silico methods for prediction of coiled-coil domains in proteins.

    PubMed

    Li, Chen; Ching Han Chang, Catherine; Nagel, Jeremy; Porebski, Benjamin T; Hayashida, Morihiro; Akutsu, Tatsuya; Song, Jiangning; Buckle, Ashley M

    2016-03-01

    Coiled-coils refer to a bundle of helices coiled together like strands of a rope. It has been estimated that nearly 3% of protein-encoding regions of genes harbour coiled-coil domains (CCDs). Experimental studies have confirmed that CCDs play a fundamental role in subcellular infrastructure and controlling trafficking of eukaryotic cells. Given the importance of coiled-coils, multiple bioinformatics tools have been developed to facilitate the systematic and high-throughput prediction of CCDs in proteins. In this article, we review and compare 12 sequence-based bioinformatics approaches and tools for coiled-coil prediction. These approaches can be categorized into two classes: coiled-coil detection and coiled-coil oligomeric state prediction. We evaluated and compared these methods in terms of their input/output, algorithm, prediction performance, validation methods and software utility. All the independent testing data sets are available at http://lightning.med.monash.edu/coiledcoil/. In addition, we conducted a case study of nine human polyglutamine (PolyQ) disease-related proteins and predicted CCDs and oligomeric states using various predictors. Prediction results for CCDs were highly variable among different predictors. Only two peptides from two proteins were confirmed to be CCDs by majority voting. Both domains were predicted to form dimeric coiled-coils using oligomeric state prediction. We anticipate that this comprehensive analysis will be an insightful resource for structural biologists with limited prior experience in bioinformatics tools, and for bioinformaticians who are interested in designing novel approaches for coiled-coil and its oligomeric state prediction.

  2. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  3. Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains.

    PubMed

    Wang, C; Stewart, R J; Kopecek, J

    1999-02-04

    Stimuli-sensitive polymer hydrogels, which swell or shrink in response to changes in the environmental conditions, have been extensively investigated and used as 'smart' biomaterials and drug-delivery systems. Most of these responsive hydrogels are prepared from a limited number of synthetic polymers and their derivatives, such as copolymers of (meth)acrylic acid, acrylamide and N-isopropyl acrylamide. Water-soluble synthetic polymers have also been crosslinked with molecules of biological origin, such as oligopeptides and oligodeoxyribonucleotides, or with intact native proteins. Very often there are several factors influencing the relationship between structure and properties in these systems, making it difficult to engineer hydrogels with specified responses to particular stimuli. Here we report a hybrid hydrogel system assembled from water-soluble synthetic polymers and a well-defined protein-folding motif, the coiled coil. These hydrogels undergo temperature-induced collapse owing to the cooperative conformational transition of the coiled-coil protein domain. This system shows that well-characterized water-soluble synthetic polymers can be combined with well-defined folding motifs of proteins in hydrogels with engineered volume-change properties.

  4. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins.

    PubMed

    Caillat, Christophe; Fish, Alexander; Pefani, Dafni Eleftheria; Taraviras, Stavros; Lygerou, Zoi; Perrakis, Anastassis

    2015-11-01

    GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin-Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  5. Routine phasing of coiled-coil protein crystal structures with AMPLE

    PubMed Central

    Thomas, Jens M. H.; Keegan, Ronan M.; Bibby, Jaclyn; Winn, Martyn D.; Mayans, Olga; Rigden, Daniel J.

    2015-01-01

    Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallo­graphic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing. PMID:25866657

  6. Noncationic Rigid and Anisotropic Coiled-Coil Proteins Exhibit Cell-Penetration Activity.

    PubMed

    Nakayama, Norihisa; Hagiwara, Kyoji; Ito, Yoshihiro; Ijiro, Kuniharu; Osada, Yoshihito; Sano, Ken-ichi

    2015-08-04

    Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. However, cationic molecules tend to be cytotoxic and cause inflammation, and their stability in the blood is usually low. We have previously demonstrated that a rigid and fibrous cationic coiled-coil protein exhibited cell-penetrating ability superior to that of previously reported cell-penetrating peptides. Making use of structural properties, here we describe the cell-penetrating activity of a rigid and fibrous coiled-coil protein with a noncationic surface. A fibrous coiled-coil protein of pI 6.5 penetrated 100% of the cells tested in vitro at a concentration of 500 nM, which is comparable to that of previously reported cell-penetrating peptides. We also investigated the effect of cell-strain dependency and short-term cytotoxicity.

  7. Automated de novo phasing and model building of coiled-coil proteins.

    PubMed

    Rämisch, Sebastian; Lizatović, Robert; André, Ingemar

    2015-03-01

    Models generated by de novo structure prediction can be very useful starting points for molecular replacement for systems where suitable structural homologues cannot be readily identified. Protein-protein complexes and de novo-designed proteins are examples of systems that can be challenging to phase. In this study, the potential of de novo models of protein complexes for use as starting points for molecular replacement is investigated. The approach is demonstrated using homomeric coiled-coil proteins, which are excellent model systems for oligomeric systems. Despite the stereotypical fold of coiled coils, initial phase estimation can be difficult and many structures have to be solved with experimental phasing. A method was developed for automatic structure determination of homomeric coiled coils from X-ray diffraction data. In a benchmark set of 24 coiled coils, ranging from dimers to pentamers with resolutions down to 2.5 Å, 22 systems were automatically solved, 11 of which had previously been solved by experimental phasing. The generated models contained 71-103% of the residues present in the deposited structures, had the correct sequence and had free R values that deviated on average by 0.01 from those of the respective reference structures. The electron-density maps were of sufficient quality that only minor manual editing was necessary to produce final structures. The method, named CCsolve, combines methods for de novo structure prediction, initial phase estimation and automated model building into one pipeline. CCsolve is robust against errors in the initial models and can readily be modified to make use of alternative crystallographic software. The results demonstrate the feasibility of de novo phasing of protein-protein complexes, an approach that could also be employed for other small systems beyond coiled coils.

  8. Isolation and cloning of Omp alpha, a coiled-coil protein spanning the periplasmic space of the ancestral eubacterium Thermotoga maritima.

    PubMed Central

    Engel, A M; Cejka, Z; Lupas, A; Lottspeich, F; Baumeister, W

    1992-01-01

    We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils. Images PMID:1330536

  9. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil protein assemblies

    PubMed Central

    Wood, Christopher W.; Bruning, Marc; Ibarra, Amaurys Á.; Bartlett, Gail J.; Thomson, Andrew R.; Sessions, Richard B.; Brady, R Leo; Woolfson, Derek N.

    2014-01-01

    Motivation: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. Results: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. Availability and implementation: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/ Contact: D.N.Woolfson@bristol.ac.uk or Chris.Wood@bristol.ac.uk PMID:25064570

  10. The Symmetrical Structure of Structural Maintenance of Chromosomes (SMC) and MukB Proteins: Long, Antiparallel Coiled Coils, Folded at a Flexible Hinge

    PubMed Central

    Melby, Thomas E.; Ciampaglio, Charles N.; Briscoe, Gina; Erickson, Harold P.

    1998-01-01

    Structural maintenance of chromosomes (SMC) proteins function in chromosome condensation and several other aspects of DNA processing. They are large proteins characterized by an NH2-terminal nucleotide triphosphate (NTP)-binding domain, two long segments of coiled coil separated by a hinge, and a COOH-terminal domain. Here, we have visualized by EM the SMC protein from Bacillus subtilis (BsSMC) and MukB from Escherichia coli, which we argue is a divergent SMC protein. Both BsSMC and MukB show two thin rods with globular domains at the ends emerging from the hinge. The hinge appears to be quite flexible: the arms can open up to 180°, separating the terminal domains by 100 nm, or close to near 0°, bringing the terminal globular domains together. A surprising observation is that the ∼300–amino acid–long coiled coils are in an antiparallel arrangement. Known coiled coils are almost all parallel, and the longest antiparallel coiled coils known previously are 35–45 amino acids long. This antiparallel arrangement produces a symmetrical molecule with both an NH2- and a COOH-terminal domain at each end. The SMC molecule therefore has two complete and identical functional domains at the ends of the long arms. The bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the SMC proteins. PMID:9744887

  11. Protein-based hydrogels self-assembled from genetically engineered triblock polypeptides containing coiled-coil domains

    NASA Astrophysics Data System (ADS)

    Xu, Chunyu

    Protein-based biomaterials have great potential in biomedical applications due to their similar composition with biological organisms. Environment-sensitive hydrogels based on proteins can undergo sol-gel transition due to the conformational change of the proteins in response to external stimuli. The physical properties of these hydrogels can be tailored by modification of the protein structures. Two major hypotheses were made in this dissertation. One was that coiled-coil folding motifs could be a good candidate for physical crosslinking in protein-based hydrogels, and the other was that the conformational change of coiled-coils in response to external stimuli could mediate the sol-gel transition of the protein-based hydrogels. The first part established synthesis strategies of the coiled-coil containing proteins using a genetic engineering technique. An important observation was made that the fusion sequence on the proteins could influence the thermal stability of the proteins. In the second part of the research, the self-assembly of hydrogels from a series of triblock polypeptides containing coiled-coils was evaluated. It was found that the hydrogels had a porous interconnected network microstructure. The hydrogels responded to temperature and pH, which correlated to the temperature- and pH-triggered structural transition of the coiled-coil domains. In addition, the formation of hydrogels was reversible in the present or absence of guanidine hydrochloride (GdnHCl). The last part of the research attempted to explore the relationship between the structure of the protein polymers and the physical property of the hydrogels, and to investigate the parameters influencing the hydrogel formation and physical properties. Triblock and diblock polypeptides were designed to contain different lengths of coiled-coil domains. Tyrosine residues were incorporated at selected solvent-exposed positions in order to increase the hydrophobicity of the coiled-coil domains. The

  12. The promotion of angiogenesis by growth factors integrated with ECM proteins through coiled-coil structures.

    PubMed

    Assal, Yasmine; Mie, Masayasu; Kobatake, Eiry

    2013-04-01

    An appropriate method to bind extracellular matrix (ECM) proteins and growth factors using advanced protein engineering techniques has the potential to enhance cell proliferation and differentiation for tissue regeneration and repair. In this study we developed a method to co-immobilize non-covalently an ECM protein to three different types of growth factors: basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and single-chain vascular endothelial growth factor (scVEGF121) through a coiled-coil structure formed by helixA/helixB in order to promote angiogenesis. The designed ECM was established by fusing two repeats of elastin-derived unit (APGVGV)(12), cell-adhesive sequence (RGD), laminin-derived IKVAV sequence and collagen-binding domain (CBD) to obtain CBDEREI2. HelixA was fused to each growth factor and helixB to the engineered ECM. Human umbilical vein endothelial cells (HUVECs) were cultured on engineered ECM and growth factors connected through the coiled-coil formation between helixA and helixB. Cell proliferation and capillary tube-like formation were monitored. Moreover, the differentiated cells with high expression of Ang-2 suggested the ECM remodeling. Our approach of non-covalent coupling method should provide a protein-release control system as a new contribution in biomaterial for tissue engineering field.

  13. Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles

    PubMed Central

    Indelicato, Giuliana; Wahome, Newton; Ringler, Philippe; Müller, Shirley A.; Nieh, Mu-Ping; Burkhard, Peter; Twarock, Reidun

    2016-01-01

    Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications. PMID:26840729

  14. Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.

    PubMed

    Indelicato, Giuliana; Wahome, Newton; Ringler, Philippe; Müller, Shirley A; Nieh, Mu-Ping; Burkhard, Peter; Twarock, Reidun

    2016-02-02

    Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.

  15. De novo designed coiled-coil proteins with variable conformations as components of molecular electronic devices.

    PubMed

    Shlizerman, Clara; Atanassov, Alexander; Berkovich, Inbal; Ashkenasy, Gonen; Ashkenasy, Nurit

    2010-04-14

    Conformational changes of proteins are widely used in nature for controlling cellular functions, including ligand binding, oligomerization, and catalysis. Despite the fact that different proteins and artificial peptides have been utilized as electron-transfer mediators in electronic devices, the unique propensity of proteins to switch between different conformations has not been used as a mechanism to control device properties and performance. Toward this aim, we have designed and prepared new dimeric coiled-coil proteins that adopt different conformations due to parallel or antiparallel relative orientations of their monomers. We show here that controlling the conformation of these proteins attached as monolayers to gold, which dictates the direction and magnitude of the molecular dipole relative to the surface, results in quantitative modulation of the gold work function. Furthermore, charge transport through the proteins as molecular bridges is controlled by the different protein conformations, producing either rectifying or ohmic-like behavior.

  16. Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima.

    PubMed

    Miserez, Ali; Li, Youli; Cagnon, Joel; Weaver, James C; Waite, J Herbert

    2012-02-13

    An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.

  17. A coiled coil switch mediates cold sensing by the thermosensory protein DesK.

    PubMed

    Saita, Emilio; Abriata, Luciano A; Tsai, Yi Ting; Trajtenberg, Felipe; Lemmin, Thomas; Buschiazzo, Alejandro; Dal Peraro, Matteo; de Mendoza, Diego; Albanesi, Daniela

    2015-10-01

    The thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.

  18. Insights on the structure and stability of Licanantase: a trimeric acid-stable coiled-coil lipoprotein from Acidithiobacillus thiooxidans.

    PubMed

    Abarca, Fernando; Gutierrez-Maldonado, Sebastian E; Parada, Pilar; Martinez, Patricio; Maass, Alejandro; Perez-Acle, Tomas

    2014-01-01

    Licanantase (Lic) is the major component of the secretome of Acidithiobacillus thiooxidans when grown in elemental sulphur. When used as an additive, Lic improves copper recovery from bioleaching processes. However, this recovery enhancement is not fully understood. In this context, our aim is to predict the 3D structure of Lic, to shed light on its structure-function relationships. Bioinformatics analyses on the amino acid sequence of Lic showed a great similarity with Lpp, an Escherichia coli Lipoprotein that can form stable trimers in solution. Lic and Lpp share the secretion motif, intracellular processing and alpha helix structure, as well as the distribution of hydrophobic residues in heptads forming a hydrophobic core, typical of coiled-coil structures. Cross-linking experiments showed the presence of Lic trimers, supporting our predictions. Taking the in vitro and in silico evidence as a whole, we propose that the most probable structure for Lic is a trimeric coiled-coil. According to this prediction, a suitable model for Lic was produced using the de novo algorithm "Rosetta Fold-and-Dock". To assess the structural stability of our model, Molecular Dynamics (MD) and Replica Exchange MD simulations were performed using the structure of Lpp and a 14-alanine Lpp mutant as controls, at both acidic and neutral pH. Our results suggest that Lic was the most stable structure among the studied proteins in both pH conditions. This increased stability can be explained by a higher number of both intermonomer hydrophobic contacts and hydrogen bonds, key elements for the stability of Lic's secondary and tertiary structure.

  19. Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation

    PubMed Central

    Behrens, Caroline; Binotti, Beyenech; Schmidt, Carla; Robinson, Carol V.; Chua, John Jia En; Kühnel, Karin

    2013-01-01

    The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction. PMID:24098481

  20. Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction

    PubMed Central

    Stewart, Chelsea M.; Buffalo, Cosmo Z.; Valderrama, J. Andrés; Henningham, Anna; Cole, Jason N.; Nizet, Victor; Ghosh, Partho

    2016-01-01

    The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A “capture-and-collapse” model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis. PMID:27512043

  1. A family of small coiled-coil-forming proteins functioning at the late endosome in yeast.

    PubMed

    Kranz, A; Kinner, A; Kölling, R

    2001-03-01

    The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

  2. Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

    PubMed

    Texada, Michael J; Simonette, Rebecca A; Deery, William J; Beckingham, Kathleen M

    2011-02-15

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions.

  3. TROPOMYOSIN IS AN INTERACTION PARTNER OF THE DROSOPHILA COILED COIL PROTEIN YURI GAGARIN

    PubMed Central

    Texada, Michael J.; Simonette, Rebecca A.; Deery, William J.; Beckingham, Kathleen M.

    2011-01-01

    The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin “cones” that mediate spermatid individualization. We used tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuriF64, failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions. PMID:21126519

  4. Coiled Coil Rich Proteins (Ccrp) Influence Molecular Pathogenicity of Helicobacter pylori

    PubMed Central

    Schätzle, Sarah; Specht, Mara; Waidner, Barbara

    2015-01-01

    Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak “scattering/hummingbird” phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen. PMID:25822999

  5. Coiled Coil Domain-containing Protein 56 (CCDC56) Is a Novel Mitochondrial Protein Essential for Cytochrome c Oxidase Function*

    PubMed Central

    Peralta, Susana; Clemente, Paula; Sánchez-Martínez, Álvaro; Calleja, Manuel; Hernández-Sierra, Rosana; Matsushima, Yuichi; Adán, Cristina; Ugalde, Cristina; Fernández-Moreno, Miguel Ángel; Kaguni, Laurie S.; Garesse, Rafael

    2012-01-01

    In Drosophila melanogaster, the mitochondrial transcription factor B1 (d-mtTFB1) transcript contains in its 5′-untranslated region a conserved upstream open reading frame denoted as CG42630 in FlyBase. We demonstrate that CG42630 encodes a novel protein, the coiled coil domain-containing protein 56 (CCDC56), conserved in metazoans. We show that Drosophila CCDC56 protein localizes to mitochondria and contains 87 amino acids in flies and 106 in humans with the two proteins sharing 42% amino acid identity. We show by rapid amplification of cDNA ends and Northern blotting that Drosophila CCDC56 protein and mtTFB1 are encoded on a bona fide bicistronic transcript. We report the generation and characterization of two ccdc56 knock-out lines in Drosophila carrying the ccdc56D6 and ccdc56D11 alleles. Lack of the CCDC56 protein in flies induces a developmental delay and 100% lethality by arrest of larval development at the third instar. ccdc56 knock-out larvae show a significant decrease in the level of fully assembled cytochrome c oxidase (COX) and in its activity, suggesting a defect in complex assembly; the activity of the other oxidative phosphorylation complexes remained either unaffected or increased in the ccdc56 knock-out larvae. The lethal phenotype and the decrease in COX were partially rescued by reintroduction of a wild-type UAS-ccdc56 transgene. These results indicate an important role for CCDC56 in the oxidative phosphorylation system and in particular in COX function required for proper development in D. melanogaster. We propose CCDC56 as a candidate factor required for COX biogenesis/assembly. PMID:22610097

  6. Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration

    PubMed Central

    Kelsey, Jessica S.; Fastman, Nathan M.; Noratel, Elizabeth F.; Blumberg, Daphne D.

    2012-01-01

    The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain—a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein–expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm− cells. During migration ndm− cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function—cell spreading—was also defective in the ndm− cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm− cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures. PMID:22809629

  7. Allosteric effects in coiled-coil proteins folding and lanthanide-ion binding.

    PubMed

    Samiappan, Manickasundaram; Alasibi, Samaa; Cohen-Luria, Rivka; Shanzer, Abraham; Ashkenasy, Gonen

    2012-10-07

    Peptide sequences modified with lanthanide-chelating groups at their N-termini, or at their lysine side chains, were synthesized, and new Ln(III) complexes were characterized. We show that partial folding of the conjugates to form trimer coiled coil structures induces coordination of lanthanides to the ligand, which in turn further stabilizes the 3D structure.

  8. The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding.

    PubMed

    Kraatz, Sebastian; Guichard, Paul; Obbineni, Jagan M; Olieric, Natacha; Hatzopoulos, Georgios N; Hilbert, Manuel; Sen, Indrani; Missimer, John; Gönczy, Pierre; Steinmetz, Michel O

    2016-07-20

    Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.

  9. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    PubMed

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans.

  10. Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

    PubMed

    Arakawa, Takeshi; Harakuni, Tetsuya

    2014-09-03

    To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.

  11. Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

    PubMed

    Nishida, Y; Shima, F; Sen, H; Tanaka, Y; Yanagihara, C; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    1998-10-23

    In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

  12. Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.

    PubMed

    Scherer, Myriam; Klingl, Stefan; Sevvana, Madhumati; Otto, Victoria; Schilling, Eva-Maria; Stump, Joachim D; Müller, Regina; Reuter, Nina; Sticht, Heinrich; Muller, Yves A; Stamminger, Thomas

    2014-11-01

    PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to

  13. Methods for Solving Highly Symmetric de Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three Stranded Coiled Coil structure containing D-amino Acids

    PubMed Central

    Ruckthong, Leela; Stuckey, Jeanne A.; Pecoraro, Vincent L.

    2017-01-01

    The core objective of de Novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proved that a four heptad repeat scaffold contained in the three stranded coiled coil (3SCC), called CoilSer, provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a D-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CoilSer sequence, yielding a GRAND-CoilSer to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled coil designs. PMID:27586331

  14. Coiled-Coil Design: Updated and Upgraded.

    PubMed

    Woolfson, Derek N

    2017-01-01

    α-Helical coiled coils are ubiquitous protein-folding and protein-interaction domains in which two or more α-helical chains come together to form bundles. Through a combination of bioinformatics analysis of many thousands of natural coiled-coil sequences and structures, plus empirical protein engineering and design studies, there is now a deep understanding of the sequence-to-structure relationships for this class of protein architecture. This has led to considerable success in rational design and what might be termed in biro de novo design of simple coiled coils, which include homo- and hetero-meric parallel dimers, trimers and tetramers. In turn, these provide a toolkit for directing the assembly of both natural proteins and more complex designs in protein engineering, materials science and synthetic biology. Moving on, the increased and improved use of computational design is allowing access to coiled-coil structures that are rare or even not observed in nature, for example α-helical barrels, which comprise five or more α-helices and have central channels into which different functions may be ported. This chapter reviews all of these advances, outlining improvements in our knowledge of the fundamentals of coiled-coil folding and assembly, and highlighting new coiled coil-based materials and applications that this new understanding is opening up. Despite considerable progress, however, challenges remain in coiled-coil design, and the next decade promises to be as productive and exciting as the last.

  15. AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

    PubMed Central

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-01-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro. PMID:15342587

  16. AglZ is a filament-forming coiled-coil protein required for adventurous gliding motility of Myxococcus xanthus.

    PubMed

    Yang, Ruifeng; Bartle, Sarah; Otto, Rebecca; Stassinopoulos, Angela; Rogers, Matthew; Plamann, Lynda; Hartzell, Patricia

    2004-09-01

    The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

  17. The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle

    PubMed Central

    Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei; McMurry, Jonathan L.; Wilson, Kristy J.; Kwon, Grace E.; Stanford, Rachel; Deehan, Kevin; Tinley, Tina L.; Ngwa, Verra M.; Benian, Guy M.

    2016-01-01

    UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89’s SH3 domain and residues 294–376 of paramyosin and has a KD of ∼1.1 μM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89’s SH3 is α-helical and lacks prolines. Homology modeling of UNC-89’s SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a “skip residue,” which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity. PMID:27009202

  18. A Parallel Coiled-Coil Tetramer with Offset Helices

    SciTech Connect

    Liu,J.; Deng, Y.; Zheng, Q.; Cheng, C.; Kallenbach, N.; Lu, M.

    2006-01-01

    Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between {alpha} helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of {alpha}-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete {alpha}-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 {angstrom} resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

  19. Design of beta-domain swapping, alpha/beta-protein, environmentally sensitive coiled coil and peptide functionalized titania materials

    NASA Astrophysics Data System (ADS)

    Nagarkar, Radhika P.

    2009-12-01

    The objective of this dissertation is to apply rational peptide design to fabricate nanomaterials via self-assembly. This has been demonstrated in structurally diverse systems with an aim of deciphering the underlying principles governing how sequence affects the peptide's ability to adopt a specific secondary structure and ultimate material properties that are realized from the association of these secondary structural elements. Several amyloidogenic proteins have been shown to self-assemble into fibrils using a mechanism known as domain swapping. Here, discreet units of secondary structure are exchanged among discreet proteins during self-assembly to form extended networks with precise three dimensional organization. The possibility of using these mechanisms to design peptides capable of controlled assembly and fibril formation leading to materials with targeted properties is explored. By altering the placement of a beta-turn sequence that varies the size and location of the exchanged strand, twisting, non-twisting and laminated fibrillar nanostructures are obtained. Hydrogels prepared from these strand swapping beta-hairpins have varied rheological properties due to differences in their fibrillar nanostructures. In a second distinct design, alpha/beta-proteins are used to prepare environmentally sensitive hydrogels. Here, multiple distinct motifs for structural integrity and dynamic response within a single self-assembling peptide allow the amyloid-like fibrils formed to controllably alter their nano-topography in response to an external stimulus such as temperature. The development of these self-assembling alpha/beta-protein motifs also necessitated the design of pH sensitive antiparallel coiled coils. Exploring the basic principles responsible for pH dependent conformational changes in coiled coils can lead to new insights in the control of protein structure and function. Lastly, this dissertation discusses the interface between biomolecules and inorganic

  20. VSGdb: a database for trypanosome variant surface glycoproteins, a large and diverse family of coiled coil proteins

    PubMed Central

    Marcello, Lucio; Menon, Suraj; Ward, Pauline; Wilkes, Jonathan M; Jones, Nicola G; Carrington, Mark; Barry, J David

    2007-01-01

    Background Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long α-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for α helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences. Description VSGdb contains fully annotated VSG sequences from the genome sequencing project, with which it shares all identifiers and annotation, and other available sequences. The database can be queried in various ways. Sequence retrieval, in FASTA format, can deliver protein or nucleotide sequence filtered by chromosomes or contigs, gene type (functional, pseudogene, etc.), domain and domain sequence family. Retrieved sequences can be stored as a temporary database for BLAST querying, reports from which include hyperlinks to the genome project database (GeneDB) CDS Info and to individual VSGdb

  1. A procedure for refining a coiled coil protein structure using x-ray fiber diffraction and modeling.

    PubMed Central

    Briki, Fatma; Doucet, Jean; Etchebest, Catherine

    2002-01-01

    We describe a combined use of experimental and simulation techniques to configure side chains in a coiled coil structure. As already demonstrated in a previous work, x-ray diffraction patterns from hard alpha-keratin fibers in the 5.15 A meridian zone reflect the global configuration of the chi(1) dihedral angle of the coiled coil side chains. Molecular simulations, such as energy minimization and molecular dynamics, and rotameric representation in the PDB, are used here on a heterodimeric coiled coil to investigate the dihedral angle distribution along the sequence. Different procedures have been used to build the structure, the quality assessment was based on the agreement between the simulated diffraction patterns and the experimental ones in the fingerprint region of coiled coils (5.15 A). The best one for building a realistic coiled coil structure consists of placing the side chains using molecular dynamics (MD) simulations, followed by side chain positioning using SMD or SCWRL procedures. The side chains and the backbone are equilibrated during the MD until they reach an equilibrium state for the t/g(+) ratio. Positioning the side chains on the resulting backbone, using the above procedures, gives rise to a well-defined 5.15 A meridian reflection. PMID:12324400

  2. Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

    PubMed Central

    Liu, Yajun; Lee, I-Ju; Sun, Mingzhai; Lower, Casey A.; Runge, Kurt W.; Ma, Jianjie; Wu, Jian-Qiu

    2016-01-01

    Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast. PMID:27385337

  3. Mcp4, a Meiotic Coiled-Coil Protein, Plays a Role in F-Actin Positioning during Schizosaccharomyces pombe Meiosis▿

    PubMed Central

    Ohtaka, Ayami; Okuzaki, Daisuke; Saito, Takamune T.; Nojima, Hiroshi

    2007-01-01

    Some meiosis-specific proteins of Schizosaccharomyces pombe harbor coiled-coil motifs and play essential roles in meiotic progression. Here we describe Mcp4, a novel meiosis-specific protein whose expression is abruptly induced at the horsetail phase and which remains expressed until sporulation is finished. Fluorescence microscopic analysis revealed that Mcp4 alters its subcellular localization during meiosis in a manner that partially resembles the movement of F-actin during meiosis. Mcp4 and F-actin never colocalize; rather, they are located in a side-by-side manner. When forespore membrane formation begins at metaphase II, the Mcp4 signals assemble at the lagging face of the dividing nuclei. At this stage, they are sandwiched between F-actin and the nucleus. Mcp4, in turn, appears to sandwich F-actin with Meu14. In mcp4Δ cells at anaphase II, the F-actin, which is normally dumbbell-shaped, adopts an abnormal balloon shape. Spores of mcp4Δ cells were sensitive to NaCl, although their shape and viability were normal. Taken together, we conclude that Mcp4 plays a role in the accurate positioning of F-actin during S. pombe meiosis. PMID:17435009

  4. Heteronuclear NMR assignments and secondary structure of the coiled coil trimerization domain from cartilage matrix protein in oxidized and reduced forms.

    PubMed Central

    Wiltscheck, R.; Kammerer, R. A.; Dames, S. A.; Schulthess, T.; Blommers, M. J.; Engel, J.; Alexandrescu, A. T.

    1997-01-01

    The C-terminal oligomerization domain of chicken cartilage matrix protein is a trimeric coiled coil comprised of three identical 43-residue chains. NMR spectra of the protein show equivalent magnetic environments for each monomer, indicating a parallel coiled coil structure with complete threefold symmetry. Sequence-specific assignments for 1H-, 15N-, and 13C-NMR resonances have been obtained from 2D 1H NOESY and TOCSY spectra, and from 3D HNCA, 15N NOESY-HSQC, and HCCH-TOCSY spectra. A stretch of alpha-helix encompassing five heptad repeats (35 residues) has been identified from intra-chain HN-HN and HN-H alpha NOE connectivities. 3JHNH alpha coupling constants, and chemical shift indices. The alpha-helix begins immediately downstream of inter-chain disulfide bonds between residues Cys 5 and Cys 7, and extends to near the C-terminus of the molecule. The threefold symmetry of the molecule is maintained when the inter-chain disulfide bonds that flank the N-terminus of the coiled coil are reduced. Residues Ile 21 through Glu 36 show conserved chemical shifts and NOE connectivities, as well as strong protection from solvent exchange in the oxidized and reduced forms of the protein. By contrast, residues Ile 10 through Val 17 show pronounced chemical shift differences between the oxidized and reduced protein. Strong chemical exchange NOEs between HN resonances and water indicate solvent exchange on time scales faster than 10 s, and suggests a dynamic fraying of the N-terminus of the coiled coil upon reduction of the disulfide bonds. Possible roles for the disulfide crosslinks of the oligomerization domain in the function of cartilage matrix protein are proposed. PMID:9260286

  5. cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.

    PubMed

    Heckel, D; Brass, N; Fischer, U; Blin, N; Steudel, I; Türeci, O; Fackler, O; Zang, K D; Meese, E

    1997-11-01

    There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.

  6. Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Kong, Sam-Geun; Wada, Masamitsu

    2010-11-09

    Chloroplasts move toward weak light (accumulation response) and away from strong light (avoidance response). The fast and accurate movement of chloroplasts in response to ambient light conditions is essential for efficient photosynthesis and photodamage prevention in chloroplasts. Here, we report that two Arabidopsis mutants, weak chloroplast movement under blue light 1 (web1) and web2, are defective in both the avoidance and the accumulation responses. Map-based cloning revealed that both genes encode coiled-coil proteins and that WEB2 is identical to the plastid movement impaired 2 (PMI2) gene. The velocities of chloroplast movement in web1 and pmi2 were approximately threefold lower than that in the wild type. Defects in the avoidance response of web1 and pmi2 were suppressed by mutation of the J-domain protein required for chloroplast accumulation response 1 (JAC1) gene, which is essential for the accumulation response; these results indicate that WEB1 and PMI2 play a role in suppressing JAC1 under strong light conditions. A yeast two-hybrid analysis and a nuclear recruitment assay identified a physical interaction between WEB1 and PMI2, and transient expression analysis of CFP-WEB1 and YFP-PMI2 revealed that they colocalized in the cytosol. Bimolecular fluorescence complementation analysis confirmed the interaction of these proteins in the cytosol. Blue light-induced changes in short chloroplast actin filaments (cp-actin filaments) were impaired in both web1 and pmi2. Our findings suggest that a cytosolic WEB1-PMI2 complex maintains the velocity of chloroplast photorelocation movement via cp-actin filament regulation.

  7. gpwac of the T4-Type Bacteriophages: Structure, Function, and Evolution of a Segmented Coiled-Coil Protein That Controls Viral Infectivity

    PubMed Central

    Letarov, A.; Manival, X.; Desplats, C.; Krisch, H. M.

    2005-01-01

    The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the ∼50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins. PMID:15659683

  8. quick-to-court, a Drosophila mutant with elevated levels of sexual behavior, is defective in a predicted coiled-coil protein.

    PubMed Central

    Gaines, P; Tompkins, L; Woodard, C T; Carlson, J R

    2000-01-01

    Remarkably little is known about the molecular mechanisms that drive sexual behavior. We have identified a new gene, quick-to-court (qtc), whose mutations cause males to show high levels of male-male courtship. qtc males also show a novel phenotype: when placed in the presence of a virgin female, they begin courtship abnormally quickly. qtc mutations are striking in their specificity, in that many aspects of male sexual behavior are normal. We have cloned the qtc gene and found that it encodes a predicted coiled-coil protein and is expressed in the olfactory organs, central nervous system, and male reproductive tract. PMID:10747058

  9. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination

    PubMed Central

    Saito, Takamune T.; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Δ cells are similar to those of meu13Δ cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Δ cells is not so conspicuous as meu13Δ cells, and no meiotic delay is observed in mcp7Δmeu13Δ cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Δ cells, whereas Meu13 becomes less stable in mcp7Δ cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. PMID:15210864

  10. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination.

    PubMed

    Saito, Takamune T; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination.

  11. Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded coiled-coil in the NuMA protein.

    PubMed Central

    Harborth, J; Weber, K; Osborn, M

    1995-01-01

    NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis. NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation. We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments. The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified. To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity. They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy. The results directly reveal the tripartate structure of NuMA. A long central rod domain is flanked by globular end domains. The rod is 207 nm long and is at least 90% alpha-helical. It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register. The NuMA protein thus forms the longest coiled-coil currently known. Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures. Images PMID:7781599

  12. The use of ion mobility mass spectrometry to assist protein design: a case study on zinc finger fold versus coiled coil interactions.

    PubMed

    Berezovskaya, Yana; Porrini, Massimiliano; Nortcliffe, Chris; Barran, Perdita E

    2015-04-21

    The dramatic conformational change in zinc fingers on binding metal ions for DNA recognition makes their structure-function behaviour an attractive target to mimic in de novo designed peptides. Mass spectrometry, with its high throughput and low sample consumption provides insight into how primary amino acid sequence can encode stable tertiary fold. We present here the use of ion mobility mass spectrometry (IM-MS) coupled with molecular dynamics (MD) simulations as a rapid analytical platform to inform de novo design efforts for peptide-metal and peptide-peptide interactions. A dual peptide-based synthetic system, ZiCop based on a zinc finger peptide motif, and a coiled coil partner peptide Pp, have been investigated. Titration mass spectrometry determines the relative binding affinities of different divalent metal ions as Zn(2+) > Co(2+) ≫ Ca(2+). With collision induced dissociation (CID), we probe complex stability, and establish that peptide-metal interactions are stronger and more 'specific' than those of peptide-peptide complexes, and the anticipated hetero-dimeric complex is more stable than the two homo-dimers. Collision cross-sections (CCS) measurements by IM-MS reveal increased stability with respect to unfolding of the metal-bound peptide over its apo-form, and further, larger collision cross sections for the hetero-dimeric forms suggest that dimeric species formed in the absence of metal are coiled coil like. MD supports these structural assignments, backed up by data from visible light absorbance measurements.

  13. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    PubMed

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  14. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO).

    PubMed

    Alborghetti, Marcos Rodrigo; Furlan, Ariane da Silva; da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  15. Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

    PubMed Central

    da Silva, Júlio César; Sforça, Maurício Luís; Honorato, Rodrigo Vargas; Granato, Daniela Campos; dos Santos Migueleti, Deivid Lucas; Neves, Jorge L.; de Oliveira, Paulo Sergio Lopes; Paes-Leme, Adriana Franco; Zeri, Ana Carolina de Mattos; de Torriani, Iris Concepcion Linares; Kobarg, Jörg

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth. PMID:24116125

  16. The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix.

    PubMed

    Vázquez, M I; Rivas, G; Cregut, D; Serrano, L; Esteban, M

    1998-12-01

    The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and

  17. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus

    PubMed Central

    Kracklauer, Martin P.; Wiora, Heather M.; Deery, William J.; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A.; Fuller, Margaret T.; Fischer, Janice A.; Beckingham, Kathleen M.

    2010-01-01

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface. PMID:20647369

  18. The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

    PubMed

    Kracklauer, Martin P; Wiora, Heather M; Deery, William J; Chen, Xin; Bolival, Benjamin; Romanowicz, Dwight; Simonette, Rebecca A; Fuller, Margaret T; Fischer, Janice A; Beckingham, Kathleen M

    2010-08-15

    Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

  19. Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

    PubMed

    Schofield, Alice V; Steel, Rohan; Bernard, Ora

    2012-12-21

    The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

  20. Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hyrdrophobic Heptad Repeat

    SciTech Connect

    Deng,Y.; Liu, J.; Zheng, Q.; Eliezer, D.; Kallenbach, N.; Lu, M.

    2006-01-01

    Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

  1. NMR Insight into Myosin-Binding Subunit Coiled Coil Structure Reveals Binding Interface with Protein Kinase G-Iα Leucine Zipper in Vascular Function.

    PubMed

    Sharma, Alok K; Birrane, Gabriel G; Anklin, Clemens; Rigby, Alan C; Alper, Seth L

    2017-03-09

    Nitrovasodilators relax vascular smooth muscle cells (VSMC) in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or leucine zipper (LZ) domain of the myosin light-chain phosphatase (MLCP) component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-Iα has remained limited. Here, we report the three-dimensional NMR solution structure of homodimeric CC MBS in which aa 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. 15N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation-enhancement (PRE) and CSP NMR guided HADDOCK modeling of the dimer-dimer interface of the hetero-tetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS-LZ PKG-Iα low-affinity heterotetrameric complex and allow re-evaluation of the role(s) of MLCP partner polypeptides in regulation of VSMC contractility.

  2. Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R).

    PubMed

    Acharya, Asha; Rishi, Vikas; Vinson, Charles

    2006-09-26

    We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.

  3. Modulation of elasticity in functionally distinct domains of the tropomyosin coiled-coil.

    PubMed

    Lakkaraju, Sirish Kaushik; Hwang, Wonmuk

    2009-03-01

    Alpha-helical coiled-coils are common protein structural motifs. Whereas vast information is available regarding their structure, folding, and stability, far less is known about their elastic properties, even though they play mechanical roles in many cases such as tropomyosin in muscle contraction or neck stalks of kinesin or myosin motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils, either globally or locally. Global bending stiffness of standard leucine zipper coiled-coils was calculated using normal mode analysis. Mutations in hydrophobic residues involved in the knob-into-hole interface between the two alpha-helices affect elasticity significantly, whereas charged side chains forming inter-helical salt bridges do not. This suggests that coiled-coils with less regular heptad periodicity may have regional variations in flexibility. We show this by the flexibility map of tropomyosin, which was constructed by a local fluctuation analysis. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Describing the coiled-coil as a twisted tape, it is generally more flexible in the splay bending than in the bending of the broad face. Actin binding sites in alpha zones show local rigidity minima. Broken core regions due to acidic residues at the hydrophobic face such as the Asp137 and the Glu218 are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm respectively. Such variation in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding.

  4. Modulation of elasticity in functionally distinct domains of the tropomyosin coiled-coil

    PubMed Central

    Lakkaraju, Sirish Kaushik; Hwang, Wonmuk

    2009-01-01

    Alpha-helical coiled-coils are common protein structural motifs. Whereas vast information is available regarding their structure, folding, and stability, far less is known about their elastic properties, even though they play mechanical roles in many cases such as tropomyosin in muscle contraction or neck stalks of kinesin or myosin motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils, either globally or locally. Global bending stiffness of standard leucine zipper coiled-coils was calculated using normal mode analysis. Mutations in hydrophobic residues involved in the knob-into-hole interface between the two α-helices affect elasticity significantly, whereas charged side chains forming inter-helical salt bridges do not. This suggests that coiled-coils with less regular heptad periodicity may have regional variations in flexibility. We show this by the flexibility map of tropomyosin, which was constructed by a local fluctuation analysis. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Describing the coiled-coil as a twisted tape, it is generally more flexible in the splay bending than in the bending of the broad face. Actin binding sites in α zones show local rigidity minima. Broken core regions due to acidic residues at the hydrophobic face such as the Asp137 and the Glu218 are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm respectively. Such variation in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. PMID:19830262

  5. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    PubMed Central

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  6. The coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein function in pathogen recognition and signaling.

    PubMed

    Rairdan, Gregory J; Collier, Sarah M; Sacco, Melanie A; Baldwin, Thomas T; Boettrich, Teresa; Moffett, Peter

    2008-03-01

    Plant genomes encode large numbers of nucleotide binding and leucine-rich repeat (NB-LRR) proteins, some of which mediate the recognition of pathogen-encoded proteins. Following recognition, the initiation of a resistance response is thought to be mediated by the domains present at the N termini of NB-LRR proteins, either a Toll and Interleukin-1 Receptor or a coiled-coil (CC) domain. In order to understand the role of the CC domain in NB-LRR function, we have undertaken a systematic structure-function analysis of the CC domain of the potato (Solanum tuberosum) CC-NB-LRR protein Rx, which confers resistance to Potato virus X. We show that the highly conserved EDVID motif of the CC domain mediates an intramolecular interaction that is dependent on several domains within the rest of the Rx protein, including the NB and LRR domains. Other conserved and nonconserved regions of the CC domain mediate the interaction with the Ran GTPase-activating protein, RanGAP2, a protein required for Rx function. Furthermore, we show that the Rx NB domain is sufficient for inducing cell death typical of hypersensitive plant resistance responses. We describe a model of CC-NB-LRR function wherein the LRR and CC domains coregulate the signaling activity of the NB domain in a recognition-specific manner.

  7. Nuclear Magnetic Resonance Structures of GCN4p Are Largely Conserved When Ion Pairs Are Disrupted at Acidic pH but Show a Relaxation of the Coiled Coil Superhelix.

    PubMed

    Kaplan, Anne R; Brady, Megan R; Maciejewski, Mark W; Kammerer, Richard A; Alexandrescu, Andrei T

    2017-03-09

    To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of (1)H-(15)N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, (13)C rotating frame T1 relaxation (T1ρ) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

  8. Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

    PubMed

    Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

    2015-07-01

    Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

  9. Identification and characterization of the Escherichia coli envC gene encoding a periplasmic coiled-coil protein with putative peptidase activity.

    PubMed

    Hara, Hiroshi; Narita, Setsuko; Karibian, Doris; Park, James T; Yamamoto, Yoshihiro; Nishimura, Yukinobu

    2002-07-02

    PM61 is a chain-forming envC strain of Escherichia coli with a leaky outer membrane. It was found to have an oversized penicillin-binding protein 3, which was the result of an IS4 insertion in the prc gene. The other properties of PM61 were caused by the envC mutation. We cloned the envC (yibP) gene and identified the mutation site, causing a single residue substitution, H366Y, in the PM61 envC allele. The gene product was predicted to be a periplasmic protein having coiled-coil structure in the N-terminal region and homology to lysostaphin in the C-terminal region. Overexpression of envC inhibited cell growth, and overexpression of the PM61 mutant allele caused cell lysis. Disruption of the chromosomal envC caused the same defects as the envC point mutation, indicating the gene is dispensable for growth but important for normal septation/separation and cell envelope integrity.

  10. Bivalent Formation 1, a plant-conserved gene, encodes an OmpH/coiled-coil motif-containing protein required for meiotic recombination in rice.

    PubMed

    Zhou, Lian; Han, Jingluan; Chen, Yuanling; Wang, Yingxiang; Liu, Yao-Guang

    2017-03-24

    Meiosis is essential for eukaryotic sexual reproduction and plant fertility. In comparison with over 80 meiotic genes identified in Arabidopsis, there are only ~30 meiotic genes characterized in rice (Oryza sativa L.). Many genes involved in the regulation of meiotic progression remain to be determined. In this study, we identified a sterile rice mutant and cloned a new meiotic gene, OsBVF1 (Bivalent Formation 1) by map-based cloning. Molecular genetics and cytological approaches were carried out to address the function of OsBVF1 in meiosis. Phylogenetic analyses were used to study the evolution of OsBVF1 and its homologs in plant species. Here we showed that the bvf1 male meiocytes were defective in formation of meiotic double strand break, thereby resulting in a failure of bivalent formation in diakinesis and unequal chromosome segregation in anaphase I. The causal gene, OsBVF1, encodes a unique OmpH/coiled-coil motif-containing protein and its homologs are highly conserved in the plant kingdom and seem to be a single-copy gene in the majority of plant species. Our study demonstrates that OsBVF1 is a novel plant-conserved factor involved in meiotic recombination in rice, providing a new insight into understanding of meiotic progression regulation.

  11. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates.

  12. Coiled-coil coactivators play a structural role mediating interactions in hypoxia-inducible factor heterodimerization.

    PubMed

    Guo, Yirui; Scheuermann, Thomas H; Partch, Carrie L; Tomchick, Diana R; Gardner, Kevin H

    2015-03-20

    The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. These findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.

  13. Coiled-coil Coactivators Play a Structural Role Mediating Interactions in Hypoxia-inducible Factor Heterodimerization*

    PubMed Central

    Guo, Yirui; Scheuermann, Thomas H.; Partch, Carrie L.; Tomchick, Diana R.; Gardner, Kevin H.

    2015-01-01

    The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor, we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. These findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated. PMID:25627682

  14. From coiled coils to small globular proteins: design of a native-like three-helix bundle.

    PubMed Central

    Bryson, J. W.; Desjarlais, J. R.; Handel, T. M.; DeGrado, W. F.

    1998-01-01

    A monomolecular native-like three-helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core. PMID:9655345

  15. The d'--d--d' vertical triad is less discriminating than the a'--a--a' vertical triad in the antiparallel coiled-coil dimer motif.

    PubMed

    Steinkruger, Jay D; Bartlett, Gail J; Hadley, Erik B; Fay, Lindsay; Woolfson, Derek N; Gellman, Samuel H

    2012-02-08

    Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.

  16. The d′--d--d′ Vertical Triad is Less Discriminating Than the a′--a--a′ Vertical Triad in the Antiparallel Coiled-coil Dimer Motif

    PubMed Central

    Steinkruger, Jay D.; Bartlett, Gail J.; Hadley, Erik B.; Fay, Lindsay; Woolfson, Derek N.; Gellman, Samuel H.

    2012-01-01

    Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called ‘knobs-into-holes’ (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exhibit significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral at a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pair-wise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well. PMID:22296518

  17. N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces.

    PubMed

    Fletcher, Jordan M; Bartlett, Gail J; Boyle, Aimee L; Danon, Jonathan J; Rush, Laura E; Lupas, Andrei N; Woolfson, Derek N

    2017-02-17

    The α-helical coiled coil is one of the best-studied protein-protein interaction motifs. As a result, sequence-to-structure relationships are available for the prediction of natural coiled-coil sequences and the de novo design of new ones. However, coiled coils adopt a wide range of oligomeric states and topologies, and our understanding of the specification of these and the discrimination between them remains incomplete. Gaps in our knowledge assume more importance as coiled coils are used increasingly to construct biomimetic systems of higher complexity; for this, coiled-coil components need to be robust, orthogonal, and transferable between contexts. Here, we explore how the polar side chain asparagine (Asn, N) is tolerated within otherwise hydrophobic helix-helix interfaces of coiled coils. The long-held view is that Asn placed at certain sites of the coiled-coil sequence repeat selects one oligomer state over others, which is rationalized by the ability of the side chain to make hydrogen bonds, or interactions with chelated ions within the coiled-coil interior of the favored state. We test this with experiments on de novo peptide sequences traditionally considered as directing parallel dimers and trimers, and more widely through bioinformatics analysis of natural coiled-coil sequences and structures. We find that when located centrally, rather than near the termini of such coiled-coil sequences, Asn does exert the anticipated oligomer-specifying influence. However, outside of these bounds, Asn is observed less frequently in the natural sequences, and the synthetic peptides are hyperthermostable and lose oligomer-state specificity. These findings highlight that not all regions of coiled-coil repeat sequences are equivalent, and that care is needed when designing coiled-coil interfaces.

  18. PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

    PubMed

    Osadnik, Hendrik; Schöpfel, Michael; Heidrich, Eyleen; Mehner, Denise; Lilie, Hauke; Parthier, Christoph; Risselada, H Jelger; Grubmüller, Helmut; Stubbs, Milton T; Brüser, Thomas

    2015-11-01

    Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains.

  19. Cross-linking reveals laminin coiled-coil architecture

    PubMed Central

    Armony, Gad; Jacob, Etai; Moran, Toot; Levin, Yishai; Mehlman, Tevie; Levy, Yaakov; Fass, Deborah

    2016-01-01

    Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins. PMID:27815530

  20. L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling

    PubMed Central

    Naufer, M. Nabuan; Callahan, Kathryn E.; Cook, Pamela R.; Perez-Gonzalez, Cesar E.; Williams, Mark C.; Furano, Anthony V.

    2016-01-01

    Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution. PMID:26673717

  1. Crystal Structure of a Super Leucine Zipper an Extended Two-Stranded Super Long Coiled Coil

    SciTech Connect

    J Diao

    2011-12-31

    Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 {angstrom} resolution. The peptide monomer shows a helix trunk with short curved N- and C-termini. In the crystal, two monomers cross in 35{sup o} and form an X-shaped dimer, and each X-shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two-stranded, parallel, super long coiled coil rather than a discrete, two-helix coiled coil of the wild-type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild-type leucine zipper, the N-terminus of the mutant has a dramatic conformational change and the C-terminus has one more residue Glu 32 determined. The mutant X-shaped dimer has a large crossing angle of 35{sup o} instead of 18{sup o} in the wild-type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self-assembling protein fibers.

  2. Visualization of an unstable coiled coil from the scallop myosin rod.

    PubMed

    Li, Yu; Brown, Jerry H; Reshetnikova, Ludmilla; Blazsek, Antal; Farkas, László; Nyitray, László; Cohen, Carolyn

    2003-07-17

    Alpha-helical coiled coils in muscle exemplify simplicity and economy of protein design: small variations in sequence lead to remarkable diversity in cellular functions. Myosin II is the key protein in muscle contraction, and the molecule's two-chain alpha-helical coiled-coil rod region--towards the carboxy terminus of the heavy chain--has unusual structural and dynamic features. The amino-terminal subfragment-2 (S2) domains of the rods can swing out from the thick filament backbone at a hinge in the coiled coil, allowing the two myosin 'heads' and their motor domains to interact with actin and generate tension. Most of the S2 rod appears to be a flexible coiled coil, but studies suggest that the structure at the N-terminal region is unstable, and unwinding or bending of the alpha-helices near the head-rod junction seems necessary for many of myosin's functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-A-resolution crystal structure of a leucine-zipper-stabilized fragment of the scallop striated-muscle myosin rod adjacent to the head-rod junction. The N-terminal 14 residues are poorly ordered; the rest of the S2 segment forms a flexible coiled coil with poorly packed core residues. The unusual absence of interhelical salt bridges here exposes apolar core atoms to solvent.

  3. Immune responses to coiled coil supramolecular biomaterials.

    PubMed

    Rudra, Jai S; Tripathi, Pulak K; Hildeman, David A; Jung, Jangwook P; Collier, Joel H

    2010-11-01

    Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response.

  4. Statistical analysis of intrahelical ionic interactions in alpha-helices and coiled coils.

    PubMed

    Meier, Markus; Burkhard, Peter

    2006-08-01

    There are many controversies concerning whether ionic interactions in alpha-helices and coiled coils actually contribute to the stabilisation and formation of these structures. Here we used a statistical approach to probe this question. We extracted unique alpha-helical and coiled coil structures from the protein database and analysed the ionic interactions between positively and negatively charged residues. The ionic interactions were categorized according to the type, spacing and order of the residues involved. Separate datasets were produced depending on the number of alpha-helices in the coiled coils and the mutual orientation of the helices. We compared the frequency of residue configurations able to form ionic interactions with their probability to form the interaction. We found a correlation between the two variables in alpha-helices, antiparallel two-stranded coiled coils and parallel two-stranded coiled coils. This indicates that some ionic interactions are indeed important for the formation and stabilisation of alpha-helices and coiled coils. We concluded that the configurations, which have simultaneously a large probability to form the ionic interaction and a frequent occurrence, are those, which have the most stabilising effect. These are the 4RE, 3ER and 4ER interactions.

  5. Control of recombination directionality by the Listeria phage A118 protein Gp44 and the coiled-coil motif of its serine integrase.

    PubMed

    Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

    2017-03-13

    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeriamonocytogenes but is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance from a Recombination Directionality Factor (RDF). We have identified and characterized the phage-encoded RDF, Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identity of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage, and in the case of Listeria monocytogenes lysogenized by A118-family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here we identify and characterize the Recombination Directionality Factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends

  6. A Set of Computationally Designed Orthogonal Antiparallel Homodimers that Expands the Synthetic Coiled-Coil Toolkit

    PubMed Central

    2015-01-01

    Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Currently, a limited number of well-characterized protein interaction components are available. Coiled-coil interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil toolkit. In this work, we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach. There are very few antiparallel homodimers described in the literature, and none have been measured for cross-reactivity. We tested the ability of the distance-dependent statistical potential DFIRE to predict orientation preferences for coiled-coil dimers of known structure. The DFIRE model was then combined with the CLASSY multistate protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils. Experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that, furthermore, did not interact with one additional previously reported antiparallel homodimer. Two designed peptides that formed higher-order structures suggest how future design protocols could be improved. The successful designs represent a significant expansion of the existing protein-interaction toolbox for molecular engineers. PMID:25337788

  7. Computational analysis of residue contributions to coiled-coil topology.

    PubMed

    Ramos, Jorge; Lazaridis, Themis

    2011-11-01

    A variety of features are thought to contribute to the oligomeric and topological specificity of coiled coils. In previous work, we examined the determinants of oligomeric state. Here, we examine the energetic basis for the tendency of six coiled-coil peptides to align their α-helices in antiparallel orientation using molecular dynamics simulations with implicit solvation (EEF1.1). We also examine the effect of mutations known to disrupt the topology of these peptides. In agreement with experiment, ARG or LYS at a or d positions were found to stabilize the antiparallel configuration. The modeling suggests that this is not due to a-a' or d-d' repulsions but due to interactions with e' and g' residues. TRP at core positions also favors the antiparallel configuration. Residues that disfavor parallel dimers, such as ILE at d, are better tolerated in, and thus favor the antiparallel configuration. Salt bridge networks were found to be more stabilizing in the antiparallel configuration for geometric reasons: antiparallel helices point amino acid side chains in opposite directions. However, the structure with the largest number of salt bridges was not always the most stable, due to desolvation and configurational entropy contributions. In tetramers, the extent of stabilization of the antiparallel topology by core residues is influenced by the e' residue on a neighboring helix. Residues at b and c positions in some cases also contribute to stabilization of antiparallel tetramers. This work provides useful rules toward the goal of designing coiled coils with a well-defined and predictable three-dimensional structure.

  8. Crystal Structure of the Central Coiled-Coil Domain from Human Liprin-[beta]2

    SciTech Connect

    Stafford, Ryan L.; Tang, Ming-Yun; Sawaya, Michael R.; Phillips, Martin L.; Bowie, James U.

    2012-02-07

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-{alpha}s and two liprin-{beta}s which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-{beta}2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-{beta}1 and liprin-{beta}2 coiled-coils were also identified. A 2.0 {angstrom} crystal structure of the central, protease-resistant core of the liprin-{beta}2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.

  9. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery

    NASA Astrophysics Data System (ADS)

    Reja, Rahi M.; Khan, Mohsina; Singh, Sumeet K.; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N.

    2016-02-01

    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils.Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic

  10. Ginseng (Panax quinquefolius) attenuates leptin-induced cardiac hypertrophy through inhibition of p115Rho guanine nucleotide exchange factor-RhoA/Rho-associated, coiled-coil containing protein kinase-dependent mitogen-activated protein kinase pathway activation.

    PubMed

    Moey, Melissa; Rajapurohitam, Venkatesh; Zeidan, Asad; Karmazyn, Morris

    2011-12-01

    Leptin is a 16-kDa peptide primarily derived from white adipocytes and is typically elevated in plasma of obese individuals. Although leptin plays a critical role in appetite regulation, leptin receptors have been identified in numerous tissues including the heart and have been shown to directly mediate cardiac hypertrophy through RhoA/ROCK (Ras homolog gene family, member A/Rho-associated, coiled-coil containing protein kinase)-dependent p38 mitogen-activated protein kinase (MAPK) activation; however, the basis for RhoA stimulation is unknown. Rho guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP resulting in Rho activation and may be the potential upstream factors mediating leptin-induced RhoA activation and therefore a potential target for inhibition. We investigated the effects of North American ginseng (Panax quinquefolius), reported to reduce cardiac hypertrophy, on RhoA/ROCK and MAPK activation in ventricular cardiomyocytes exposed to leptin (50 ng/ml) and the possible role of p115RhoGEF and p63RhoGEF in these responses. Leptin produced a robust hypertrophic response that was associated with RhoA/ROCK activation resulting in a significant increase in cofilin-2 phosphorylation and actin polymerization, the latter evidenced by a reduction in the G/F actin ratio. These effects were prevented by ginseng (10 μg/ml). The stimulation of RhoA/ROCK by leptin was associated with significantly increased p115RhoGEF gene and protein expression and exchange activity, all of which were completely prevented by ginseng. The ability of ginseng to prevent leptin-induced activation of RhoA/ROCK was further associated with diminished p38 MAPK activation and nuclear translocation. These results demonstrate a potent inhibitory effect of ginseng against leptin-induced cardiac hypertrophy, an effect associated with prevention of p115RhoGEF-RhoA/ROCK-dependent p38 MAPK activation.

  11. A high-resolution structure that provides insight into coiled-coil thiodepsipeptide dynamic chemistry.

    PubMed

    Dadon, Zehavit; Samiappan, Manickasundaram; Shahar, Anat; Zarivach, Raz; Ashkenasy, Gonen

    2013-09-16

    Stable and reactive: A crystal structure at 1.35 Å of a thioester coiled-coil protein reveals high similarity to all-peptide-bond proteins. In these assemblies, the thioester bonds are kept reactive towards thiol molecules in the mixture. This enables efficient domain exchange between proteins in response to changes in folding conditions or introduction of external templates.

  12. The intrinsic factor-vitamin B12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix.

    PubMed

    Lindblom, A; Quadt, N; Marsh, T; Aeschlimann, D; Mörgelin, M; Mann, K; Maurer, P; Paulsson, M

    1999-03-05

    A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-helix.

  13. D-Cysteine Ligands Control Metal Geometries within de Novo Designed Three-Stranded Coiled Coils.

    PubMed

    Pecoraro, Vincent Louis; Ruckthong, Leela; Peacock, Anna F A; Pascoe, Cherilyn E; Hemmingsen, Lars; Stuckey, Jeanne A

    2017-04-06

    While metal ion binding to naturally occurring L-amino acid proteins is well documented, understanding the impact of the opposite chirality (D) amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a D-configuration cysteine within a designed L-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The D-chirality does not alter the native fold, but the side-chain reorientation modifies the sterics of the metal binding pocket. L-Cys side-chains within the coiled-coil have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by x-ray crystallography that D-Cys side chains are preorganized with suitable geometry to bind such a ligand. This is confirmed by comparison of the Zn(II)Cl(CSL16DC)₃²¯ to the published Zn(II)(H₂O)(GRAND-CSL12AL16LC)₃¯.¹ Spectroscopic analysis indicates that the Cd(II) geometry observed using L-Cys ligands (a mixture of 3- and 4- coordinate Cd(II)) is altered to a single 4-coordinate specie when D-Cys is present. This work opens a new avenue for the control of metal site environment in man-made proteins, by simply altering the binding ligand with its mirror imaged D-configuration. Thus, use of D amino acids in a metal's coordination sphere promises to be a powerful tool for controlling the properties of future metalloproteins.

  14. Tubulin polymerization promoting protein 1 (Tppp1) phosphorylation by Rho-associated coiled-coil kinase (rock) and cyclin-dependent kinase 1 (Cdk1) inhibits microtubule dynamics to increase cell proliferation.

    PubMed

    Schofield, Alice V; Gamell, Cristina; Suryadinata, Randy; Sarcevic, Boris; Bernard, Ora

    2013-03-15

    Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

  15. Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus.

    PubMed

    Hillar, Alexander; Tripet, Brian; Zoetewey, David; Wood, Janet M; Hodges, Robert S; Boggs, Joan M

    2003-12-30

    Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer

  16. Baculovirus FP25K Localization: Role of the Coiled-Coil Domain.

    PubMed

    Garretson, Tyler A; McCoy, Jason C; Cheng, Xiao-Wen

    2016-11-01

    Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking

  17. Characterization of the baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain.

    PubMed

    Wilson, J A; Hill, J E; Kuzio, J; Faulkner, P

    1995-12-01

    The DNA sequence and transcription pattern of the p10 gene from the spruce budworm baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV) were analysed. The CfMNPV p10 gene codes for a protein 81 amino acids in length: this is the shortest p10 peptide identified thus far. A novel characteristic of the CfMNPV p10 gene is that it contains tandem late initiation sites (TAAG) having different temporal transcription patterns. Comparative analysis of CfMNPV p10 and the amino acid sequences of other p10 gene products showed that they each appear to have a similar N-terminal structure: an amphipathic alpha-helical terminus which condenses as coiled-coil multimers. Another feature of the p10 N terminus is that the central region of the coiled-coil domain resembles a bend or hairpin loop and could fold into a hairpin or form a bent rod structure. The condensation of p10 monomers to coiled-coil multimers is likely to be a step leading to the formation of p10 fibrous bodies in infected cells.

  18. Membrane fusion mediated by coiled coils: a hypothesis.

    PubMed Central

    Bentz, J

    2000-01-01

    A molecular model of the low-pH-induced membrane fusion by influenza hemagglutinin (HA) is proposed based upon the hypothesis that the conformational change to the extended coiled coil creates a high-energy hydrophobic membrane defect in the viral envelope or HA expressing cell. It is known that 1) an aggregate of at least eight HAs is required at the fusion site, yet only two or three of these HAs need to undergo the "essential" conformational change for the first fusion pore to form (Bentz, J. 2000. Biophys. J. 78:000-000); 2) the formation of the first fusion pore signifies a stage of restricted lipid flow into the nascent fusion site; and 3) some HAs can partially insert their fusion peptides into their own viral envelopes at low pH. This suggests that the committed step for HA-mediated fusion begins with a tightly packed aggregate of HAs whose fusion peptides are inserted into their own viral envelope, which causes restricted lateral lipid flow within the HA aggregate. The transition of two or three HAs in the center of the aggregate to the extended coiled coil extracts the fusion peptide and creates a hydrophobic defect in the outer monolayer of the virion, which is stabilized by the closely packed HAs. These HAs are inhibited from diffusing away from the site to admit lateral lipid flow, in part because that would initially increase the surface area of hydrophobic exposure. The other obvious pathway to heal this hydrophobic defect, or some descendent, is recruitment of lipids from the outer monolayer of the apposed target membrane, i.e., fusion. Other viral fusion proteins and the SNARE fusion protein complex appear to fit within this hypothesis. PMID:10653801

  19. Coiled coils and SAH domains in cytoskeletal molecular motors.

    PubMed

    Peckham, Michelle

    2011-10-01

    Cytoskeletal motors include myosins, kinesins and dyneins. Myosins move along tracks of actin filaments, whereas kinesins and dyneins move along microtubules. Many of these motors are involved in trafficking cargo in cells. However, myosins are mostly monomeric, whereas kinesins are mostly dimeric, owing to the presence of a coiled coil. Some myosins (myosins 6, 7 and 10) contain an SAH (single α-helical) domain, which was originally thought to be a coiled coil. These myosins are now known to be monomers, not dimers. The differences between SAH domains and coiled coils are described and the potential roles of SAH domains in molecular motors are discussed.

  20. The mei-P26 gene encodes a RING finger B-box coiled-coil-NHL protein that regulates seizure susceptibility in Drosophilia.

    PubMed

    Glasscock, Edward; Singhania, Ayush; Tanouye, Mark A

    2005-08-01

    Seizure-suppressor mutations provide unique insight into the genes and mechanisms involved in regulating nervous system excitability. Drosophila bang-sensitive (BS) mutants present a useful tool for identifying seizure suppressors since they are a well-characterized epilepsy model. Here we describe the isolation and characterization of a new Drosophila seizure-suppressor mutant that results from disruption of the meiotic gene mei-P26, which belongs to the RBCC-NHL family of proteins. The mei-P26 mutation reduces seizures in easily shocked (eas) and slamdance (sda) epileptic flies following mechanical stimulation and electroconvulsive shock. In addition, mutant mei-P26 flies exhibit seizure thresholds at least threefold greater than those of wild type. The mei-P26 phenotypes appear to result from missense mutation of a critical residue in the NHL protein-protein interaction domain of the protein. These results reveal a surprising role for mei-P26 outside of the germline as a regulator of seizure susceptibility, possibly by affecting synaptic development as a ubiquitin ligase.

  1. Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry

    PubMed Central

    1987-01-01

    We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right- handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled- coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three- dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose

  2. NMR assignment and secondary structure of coiled coil domain of C-terminal myosin binding subunit of myosin phosphatase.

    PubMed

    Sharma, Alok K; Rigby, Alan C

    2014-07-01

    Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

  3. Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in Saccharomyces cerevisiae.

    PubMed

    Samel, Anke; Rudner, Adam; Ehrenhofer-Murray, Ann E

    2017-04-03

    Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA(+) ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function in vivo, as well in vitro binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci HMR and HML to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins.

  4. Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in Saccharomyces cerevisiae

    PubMed Central

    Samel, Anke; Rudner, Adam; Ehrenhofer-Murray, Ann E.

    2017-01-01

    Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA+ ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function in vivo, as well in vitro binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci HMR and HML to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins. PMID:28188183

  5. An engineered right-handed coiled coil domain imparts extreme thermostability to the KcsA channel.

    PubMed

    Yuchi, Zhiguang; Pau, Victor P T; Lu, Bridget X; Junop, Murray; Yang, Daniel S C

    2009-11-01

    KcsA, a potassium channel from Streptomyces lividans, was the first ion channel to have its transmembrane domain structure determined by crystallography. Previously we have shown that its C-terminal cytoplasmic domain is crucial for the thermostability and the expression of the channel. Expression was almost abolished in its absence, but could be rescued by the presence of an artificial left-handed coiled coil tetramerization domain GCN4. In this study, we noticed that the handedness of GCN4 is not the same as the bundle crossing of KcsA. Therefore, a compatible right-handed coiled coil structure was identified from the Protein Data Bank and used to replace the C-terminal domain of KcsA. The hybrid channel exhibited a higher expression level than the wild-type and is extremely thermostable. Surprisingly, this stable hybrid channel is equally active as the wild-type channel in conducting potassium ions through a lipid bilayer at an acidic pH. We suggest that a similar engineering strategy could be applied to other ion channels for both functional and structural studies.

  6. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    NASA Astrophysics Data System (ADS)

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-04-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. movement protein | virion-associated protein | Biacore

  7. Data-Driven Prediction and Design of bZIP Coiled-Coil Interactions

    PubMed Central

    Potapov, Vladimir; Kaplan, Jenifer B.; Keating, Amy E.

    2015-01-01

    Selective dimerization of the basic-region leucine-zipper (bZIP) transcription factors presents a vivid example of how a high degree of interaction specificity can be achieved within a family of structurally similar proteins. The coiled-coil motif that mediates homo- or hetero-dimerization of the bZIP proteins has been intensively studied, and a variety of methods have been proposed to predict these interactions from sequence data. In this work, we used a large quantitative set of 4,549 bZIP coiled-coil interactions to develop a predictive model that exploits knowledge of structurally conserved residue-residue interactions in the coiled-coil motif. Our model, which expresses interaction energies as a sum of interpretable residue-pair and triplet terms, achieves a correlation with experimental binding free energies of R = 0.68 and significantly out-performs other scoring functions. To use our model in protein design applications, we devised a strategy in which synthetic peptides are built by assembling 7-residue native-protein heptad modules into new combinations. An integer linear program was used to find the optimal combination of heptads to bind selectively to a target human bZIP coiled coil, but not to target paralogs. Using this approach, we designed peptides to interact with the bZIP domains from human JUN, XBP1, ATF4 and ATF5. Testing more than 132 candidate protein complexes using a fluorescence resonance energy transfer assay confirmed the formation of tight and selective heterodimers between the designed peptides and their targets. This approach can be used to make inhibitors of native proteins, or to develop novel peptides for applications in synthetic biology or nanotechnology. PMID:25695764

  8. Coiled-Coil Irregularities and Instabilities in Group A Streptococcus M1 Are Required for Virulence

    SciTech Connect

    McNamara, Case; Zinkernagel, Annelies S.; Macheboeuf, Pauline; Cunningham, Madeleine W.; Nizet, Victor; Ghosh, Partho

    2008-07-21

    Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the -3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

  9. A De Novo Designed Coiled-Coil Peptide with a Reversible pH-Induced Oligomerization Switch.

    PubMed

    Lizatović, Robert; Aurelius, Oskar; Stenström, Olof; Drakenberg, Torbjörn; Akke, Mikael; Logan, Derek T; André, Ingemar

    2016-06-07

    Protein conformational switches have many useful applications but are difficult to design rationally. Here we demonstrate how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold. We describe a de novo designed peptide that was discovered to switch between a parallel symmetric pentamer at pH 8 and a trimer of antiparallel dimers at pH 6. The transition between pentamer and hexamer is caused by changes in the protonation states of glutamatic acid residues with highly upshifted pKa values in both oligomer forms. The drastic conformational change coupled with the narrow pH range makes the peptide sequence an attractive candidate for introduction of pH sensing into other proteins. The results highlight the remarkable ability of simple-α helices to self-assemble into a vast range of structural states.

  10. Strong contributions from vertical triads to helix-partner preferences in parallel coiled coils.

    PubMed

    Steinkruger, Jay D; Bartlett, Gail J; Woolfson, Derek N; Gellman, Samuel H

    2012-09-26

    Pairing preferences in heterodimeric coiled coils are determined by complementarities among side chains that pack against one another at the helix-helix interface. However, relationships between dimer stability and interfacial residue identity are not fully understood. In the context of the "knobs-into-holes" (KIH) packing pattern, one can identify two classes of interactions between side chains from different helices: "lateral", in which a line connecting the adjacent side chains is perpendicular to the helix axes, and "vertical", in which the connecting line is parallel to the helix axes. We have previously analyzed vertical interactions in antiparallel coiled coils and found that one type of triad constellation (a'-a-a') exerts a strong effect on pairing preferences, while the other type of triad (d'-d-d') has relatively little impact on pairing tendencies. Here, we ask whether vertical interactions (d'-a-d') influence pairing in parallel coiled-coil dimers. Our results indicate that vertical interactions can exert a substantial impact on pairing specificity, and that the influence of the d'-a-d' triad depends on the lateral a' contact within the local KIH motif. Structure-informed bioinformatic analyses of protein sequences reveal trends consistent with the thermodynamic data derived from our experimental model system in suggesting that heterotriads involving Leu and Ile are preferred over homotriads involving Leu and Ile.

  11. Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils

    NASA Astrophysics Data System (ADS)

    Kodama, Tatsuhiko; Freeman, Mason; Rohrer, Lucia; Zabrecky, James; Matsudaira, Paul; Krieger, Monty

    1990-02-01

    The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an a-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

  12. Invited review the coiled coil silk of bees, ants, and hornets.

    PubMed

    Sutherland, Tara D; Weisman, Sarah; Walker, Andrew A; Mudie, Stephen T

    2012-06-01

    In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks.

  13. ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils

    PubMed Central

    Kim, Byeong-Won; Jung, Yang Ouk; Kim, Min Kyung; Kwon, Do Hoon; Park, Si Hoon; Kim, Jun Hoe; Kuk, Yong-Boo; Oh, Sun-Joo; Kim, Leehyeon; Kim, Bong Heon; Yang, Woo Seok; Song, Hyun Kyu

    2017-01-01

    The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction. PMID:28266564

  14. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

    PubMed

    Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

    2013-06-01

    Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

  15. Peptidyl Materials Formed Through Click Chemistry Enhanced Coiled-Coil Interactions

    NASA Astrophysics Data System (ADS)

    Koehler, Kenneth

    2014-03-01

    Biologically derived materials offer a level of sophistication synthetically fabricated materials have only attempted to mimic. This level of complexity may be found in materials such as peptides. Implementing new theory and modeling, peptides with the propensity to form coiled-coil (CC) bundles were designed and synthesized. Through the use of this de novo approach, modeling allowed prediction of the feasibility to include non-natural amino acids conducive to click chemistry into the peptide. Amino acids showcasing thiol or alkyne functionalities were considered owing to the ability of these moieties to participate in the thiol-ene and copper click reactions respectively. Once synthesized, the peptides decorated with these clickable motifs were placed in solution and allowed to self-assemble into CC's. CD spectroscopy and DLS experiments confirmed the formation and assembly of CC's. Click reactions were then incited to link the CC assemblies together and form a network with predictable dimensionality and pore size between CC bundles. To incite network formation, click reactions between CC side chain residues and suitably functionalized crosslinkers were implemented. The linking of coiled-coils and material formation were assessed using DLS and TEM.

  16. Dimeric coiled-coil structure of Saccharomyces cerevisiae Atg16 and its functional significance in autophagy.

    PubMed

    Fujioka, Yuko; Noda, Nobuo N; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2010-01-08

    Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5.Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 A resolution and its CCD at 2.5 A resolution. The CCD and full-length Atg16 each exhibit an extended alpha-helix, 90 and 130 A, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.

  17. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    SciTech Connect

    Terawaki, Shin-ichi; Yoshikane, Asuka; Higuchi, Yoshiki; Wakamatsu, Kaori

    2015-05-01

    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.

  18. Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

    PubMed Central

    Rao, Tara; Ruiz-Gómez, Gloria; Hill, Timothy A.; Hoang, Huy N.; Fairlie, David P.; Mason, Jody M.

    2013-01-01

    Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases. PMID:23544065

  19. A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations

    DOE PAGES

    Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.; ...

    2015-05-06

    The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here in this paper we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms amore » well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. Finally, these novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.« less

  20. A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations

    SciTech Connect

    Malashkevich, Vladimir N.; Higgins, Chelsea D.; Almo, Steven C.; Lai, Jonathan R.

    2015-05-06

    The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here in this paper we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. Finally, these novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.

  1. Integration of Rotation and Piston Motions in Coiled-Coil Signal Transduction▿

    PubMed Central

    Gao, Rong; Lynn, David G.

    2007-01-01

    A coordinated response to a complex and dynamic environment requires an organism to simultaneously monitor and interpret multiple signaling cues. In bacteria and some eukaryotes, environmental responses depend on the histidine autokinases (HKs). For example, VirA, a large integral membrane HK from Agrobacterium tumefaciens, regulates the expression of virulence genes in response to signals from multiple molecular classes (phenol, pH, and sugar). The ability of this pathogen to perceive inputs from different known host signals within a single protein receptor provides an opportunity to understand the mechanisms of signal integration. Here we exploited the conserved domain organization of the HKs and engineered chimeric kinases to explore the signaling mechanisms of phenol sensing and pH/sugar integration. Our data implicate a piston-assisted rotation of coiled coils for integration of multiple inputs and regulation of critical responses during pathogenesis. PMID:17573470

  2. Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells.

    PubMed

    David, Ahuvit; Amartely, Hadar; Rabinowicz, Noa; Shamir, Mai; Friedler, Assaf; Izraeli, Shai

    2016-04-14

    The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes.

  3. Molecular basis of the STIL coiled coil oligomerization explains its requirement for de-novo formation of centrosomes in mammalian cells

    PubMed Central

    David, Ahuvit; Amartely, Hadar; Rabinowicz, Noa; Shamir, Mai; Friedler, Assaf; Izraeli, Shai

    2016-01-01

    The STIL protein is essential for centriole replication and for the non-templated, de novo centriole biogenesis that is required for mammalian embryogenesis. Here we performed quantitative biophysical and structural analysis of the central short coiled coil domain (CCD) of STIL that is critical for its function. Using biophysical, biochemical and cell biology approaches, we identified the specific residues in the CCD that mediate the oligomerization, centrosomal localization and protein interactions of STIL. We characterized the structural properties of the coiled coil peptide using circular dichroism spectroscopy and size exclusion chromatography. We identified two regions in this domain, containing eight hydrophobic residues, which mediate the coiled coil oligomerization. Mutations in these residues destabilized the coiled coil thermodynamically but in most cases did not affect its secondary structure. Reconstituting mouse embryonic fibroblasts lacking endogenous Stil, we show that STIL oligomerization mediated by these residues is not only important for the centrosomal functions of STIL during the canonical duplication process but also for de-novo formation of centrosomes. PMID:27075531

  4. The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

    PubMed

    Buchsbaum, R; Telliez, J B; Goonesekera, S; Feig, L A

    1996-09-01

    We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.

  5. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  6. Directed assembly of defined oligomeric photosynthetic reaction centres through adaptation with programmable extra-membrane coiled-coil interfaces.

    PubMed

    Swainsbury, David J K; Harniman, Robert L; Di Bartolo, Natalie D; Liu, Juntai; Harper, William F M; Corrie, Alexander S; Jones, Michael R

    2016-12-01

    A challenge associated with the utilisation of bioenergetic proteins in new, synthetic energy transducing systems is achieving efficient and predictable self-assembly of individual components, both natural and man-made, into a functioning macromolecular system. Despite progress with water-soluble proteins, the challenge of programming self-assembly of integral membrane proteins into non-native macromolecular architectures remains largely unexplored. In this work it is shown that the assembly of dimers, trimers or tetramers of the naturally monomeric purple bacterial reaction centre can be directed by augmentation with an α-helical peptide that self-associates into extra-membrane coiled-coil bundle. Despite this induced oligomerisation the assembled reaction centres displayed normal spectroscopic properties, implying preserved structural and functional integrity. Mixing of two reaction centres modified with mutually complementary α-helical peptides enabled the assembly of heterodimers in vitro, pointing to a generic strategy for assembling hetero-oligomeric complexes from diverse modified or synthetic components. Addition of two coiled-coil peptides per reaction centre monomer was also tolerated despite the challenge presented to the pigment-protein assembly machinery of introducing multiple self-associating sequences. These findings point to a generalised approach where oligomers or longer range assemblies of multiple light harvesting and/or redox proteins can be constructed in a manner that can be genetically-encoded, enabling the construction of new, designed bioenergetic systems in vivo or in vitro.

  7. Septin phosphorylation and coiled-coil domains function in cell and septin ring morphology in the filamentous fungus Ashbya gossypii.

    PubMed

    Meseroll, Rebecca A; Occhipinti, Patricia; Gladfelter, Amy S

    2013-02-01

    Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function.

  8. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    PubMed

    Ronzone, Erik; Paumet, Fabienne

    2013-01-01

    Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  9. Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56.

    PubMed

    Dragan, Anatoly I; Potekhin, Sergey A; Sivolob, Andrei; Lu, Min; Privalov, Peter L

    2004-11-30

    Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links

  10. Expression and characterization of transmembrane and coiled-coil domain family 3

    PubMed Central

    Sohn, Wern-Joo; Kim, Jae-Young; Kim, Dongbum; Park, Jeong-A; Lee, Younghee; Kwon, Hyung-Joo

    2016-01-01

    Transmembrane and coiled-coil domain family 3 (TMCC3) has been reported to be expressed in the human brain; however, its function is still unknown. Here, we found that expression of TMCC3 is higher in human whole brain, testis and spinal cord compared to other human tissues. TMCC3 was expressed in mouse developing hind brain, lung, kidney and somites, with strongest expression in the mesenchyme of developing tongue. By expression of recombinant TMCC3 and its deletion mutants, we found that TMCC3 proteins self-assemble to oligomerize. Immunostaining and confocal microscopy data revealed that TMCC3 proteins are localized in endoplasmic reticulum through transmembrane domains. Based on immunoprecipitation and mass spectroscopy data, TMCC3 proteins associate with TMCC3 and 14-3-3 proteins. This supports the idea that TMCC3 proteins form oligomers and that 14-3-3 may be involved in the function of TMCC3. Taken together, these results may be useful for better understanding of uncharacterized function of TMCC3. PMID:27697108

  11. SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly.

    PubMed

    Qiao, Renping; Cabral, Gabriela; Lettman, Molly M; Dammermann, Alexander; Dong, Gang

    2012-11-14

    The centriole is a conserved microtubule-based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS-5 and SAS-6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS-5 C-terminal domain (residues 390-404) specifically binds to a narrow central region (residues 275-288) of the SAS-6 coiled coil. This was supported by the crystal structure of the SAS-6 coiled-coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS-6 CCD, which gives rise to an anti-parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS-5 and SAS-6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero-complex to facilitate the correct assembly of the nine-fold symmetric centriole.

  12. Far3p domains involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in budding yeast.

    PubMed

    Lai, Fenju; Wu, Rentian; Wang, Jiafeng; Li, Chunming; Zou, Lan; Lu, Yongjun; Liang, Chun

    2011-02-01

    Far3p (factor arrest), a protein that interacts with Far7-11p, is required for the pheromone-mediated cell cycle arrest in G1 phase. We used a combination of computational and experimental strategies to identify the Far3p self-association, to map the Far3p domains that interact with Far3p itself and with other Far proteins, and to reveal the importance of the two coiled-coil motifs of Far3p in the integrity and function of the Far complex. We show that Far3p self-associates through its central region and its C-terminal coiled-coil domain, that the amino acid 61-100 region of Far3p interacts with Far7p, and that the Far3p N-terminal coiled-coil domain interacts with Far9p and Far10p. Mutation of the N-terminal coiled coil disrupts the interactions of Far3p with Far9p and Far10p, and mutation of the C-terminal domain weakens the Far3p self-interaction. Although the N- and C-terminal coiled-coil mutants reserve some of the interactions with itself and some other Far proteins, both mutants are defective in the pheromone-mediated G1 arrest, indicating that both coiled-coil motifs of Far3p are essential for the integrity and the function of the Far complex.

  13. Structure of a truncated form of leucine zipper II of JIP3 reveals an unexpected antiparallel coiled-coil arrangement.

    PubMed

    Llinas, Paola; Chenon, Mélanie; Nguyen, T Quyen; Moreira, Catia; de Régibus, Annélie; Coquard, Aline; Ramos, Maria J; Guérois, Raphaël; Fernandes, Pedro A; Ménétrey, Julie

    2016-03-01

    JIP3 and JIP4, two highly related scaffolding proteins for MAP kinases, are binding partners for two molecular motors as well as for the small G protein ARF6. The leucine zipper II (LZII) region of JIP3/4 is the binding site for these three partners. Previously, the crystal structure of ARF6 bound to JIP4 revealed LZII in a parallel coiled-coil arrangement. Here, the crystal structure of an N-terminally truncated form of LZII of JIP3 alone shows an unexpected antiparallel arrangement. Using molecular dynamics and modelling, the stability of this antiparallel LZII arrangement, as well as its specificity for ARF6, were investigated. This study highlights that N-terminal truncation of LZII can change its coiled-coil orientation without affecting its overall stability. Further, a conserved buried asparagine residue was pinpointed as a possible structural determinant for this dramatic structural rearrangement. Thus, LZII of JIP3/4 is a versatile structural motif, modifications of which can impact partner recognition and thus biological function.

  14. Central domain of DivIB caps the C-terminal regions of the FtsL/DivIC coiled-coil rod.

    PubMed

    Masson, Soizic; Kern, Thomas; Le Gouëllec, Audrey; Giustini, Cécile; Simorre, Jean-Pierre; Callow, Philip; Vernet, Thierry; Gabel, Frank; Zapun, André

    2009-10-02

    DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the "bean"-shaped central beta-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.

  15. Inhibition of the 26S proteasome by peptide mimics of the coiled-coil region of its ATPase subunits.

    PubMed

    Inobe, Tomonao; Genmei, Reiko

    Regulation of proteasomal degradation is an indispensable tool for biomedical studies. Thus, there is demand for novel proteasome inhibitors. Proteasomal degradation requires formation of coiled-coil structure by the N-terminal region of ATPase subunits of the proteasome cap. Here we show that peptides that mimic the N-terminal coiled-coil region of ATPase subunits interfere with proteasome function. These results suggest that coiled-coil peptides represent promising new proteasome inhibitors and that N-terminal coiled-coil regions of ATPase subunits are targets for proteasome inhibition.

  16. The coiled-coil domain of zebrafish TRPM7 regulates Mg·nucleotide sensitivity

    PubMed Central

    Jansen, Chad; Sahni, Jaya; Suzuki, Sayuri; Horgen, F. David; Penner, Reinhold; Fleig, Andrea

    2016-01-01

    TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel’s regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only. PMID:27628598

  17. Midbody Targeting of the ESCRT Machinery by a Noncanonical Coiled Coil in CEP55

    SciTech Connect

    Lee, Hyung Ho; Elia, Natalie; Ghirlando, Rodolfo; Lippincott-Schwartz, Jennifer; Hurley, James H.

    2008-11-14

    The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.

  18. Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly.

    PubMed

    Nepal, Manish; Sheedlo, Michael J; Das, Chittaranjan; Chmielewski, Jean

    2016-08-31

    Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design.

  19. Structural Characterization of an LPA1 Second Extracellular Loop Mimetic with a Self-Assembling Coiled-Coil Folding Constraint.

    PubMed

    Young, John K; Clayton, Benjamin T; Kikonyogo, Alexandra; Pham, Truc-Chi T; Parrill, Abby L

    2013-01-29

    G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane α-helical domains. The EL of the LPA(1) receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA(1) GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA(1)-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA(1) receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.

  20. Self-Assembly of Coiled-coil Tetramers in the 1.40 A Structure of a Leucine-zipper Mutant

    SciTech Connect

    Deng,Y.; Zheng, Q.; Liu, J.; Cheng, C.; Kallenbach, N.; Lu, M.

    2007-01-01

    The hydrophobic core of the GCN4 leucine-zipper dimerization domain is formed by a parallel helical association between nonpolar side chains at the a and d positions of the heptad repeat. Here we report a self-assembling coiled-coil array formed by the GCN4-pAe peptide that differs from the wild-type GCN4 leucine zipper by alanine substitutions at three charged e positions. GCN4-pAe is incompletely folded in normal solution conditions yet self-assembles into an antiparallel tetraplex in crystals by formation of unanticipated hydrophobic seams linking the last two heptads of two parallel doublestranded coiled coils. The GCN4-pAe tetramers in the lattice associate laterally through the identical interactions to those in the intramolecular dimer-dimer interface. The van der Waals packing interaction in the solid state controls extended supramolecular assembly of the protein, providing an unusual atomic scale view of a mesostructure.

  1. NEMO trimerizes through its coiled-coil C-terminal domain.

    PubMed

    Agou, Fabrice; Ye, Fei; Goffinont, Stéphane; Courtois, Gilles; Yamaoka, Shoji; Israël, Alain; Véron, Michel

    2002-05-17

    NEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta. To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wild-type and mutant NEMO expressed in Escherichia coli. In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex. A minor fraction of rNEMO was found tightly associated with DnaK (E. coli Hsp70). We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate. In vivo cross-linking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form. Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO. We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator.

  2. Transforming the Energy Landscape of a Coiled-Coil Peptide via Point Mutations.

    PubMed

    Röder, Konstantin; Wales, David J

    2017-03-14

    We analyze the effect of point mutations on the energy landscape of a coiled-coil peptide, GCN4-pLI, where the native state is a parallel tetrameric configuration formed from two identical dimers. Experimentally, a single mutation, E20S, supports both antiparallel and parallel structures. Here, we analyze the potential energy landscapes of the dimeric units for the parent sequence and four mutants, namely E20S, E20A, E20P, and E20G. Despite sharing characteristic funnels containing the parallel and antiparallel structures, the point mutations change some parts of the landscape quite dramatically, and we predict new intermediate structures and characterize the associated heat capacities. For the mutants we predict that kinked intermediate structures facilitate the transition between parallel and antiparallel morphologies, in contrast to the parent sequence. Furthermore, we predict a change from a multifunnel energy landscape in the E20S mutant to a landscape dominated by an underlying single funnel in the parent sequence, with accompanying heat capacity signatures. Our results imply that changes in the landscape due to mutations might provide useful tools for functional protein design.

  3. Is Five Percent Too Small? Analysis of the Overlaps between Disorder, Coiled Coil and Collagen Predictions in Complete Proteomes

    PubMed Central

    Gáspári, Zoltán

    2014-01-01

    Identification of intrinsic disorder in proteins and proteomes has revealed important novel aspects of protein function and interactions. However, it has been pointed out that several oligomeric fibrillar protein motifs such as coiled coils and collagen triple helical segments can also identified as intrinsically disordered. This feature has not yet been investigated in more detail at the proteome level. The present work aims at the identification and quantification of such overlaps in full proteomes to assess their significance in large-scale studies of protein disorder. It was found that the percentage of cross-predicted residues is around 5% in the human proteome and is generally near that value in other metazoan ones but shows remarkable variation in different organisms. In particular, smaller proteomes are increasingly prone to such cross-predictions, thus, especially the analysis of viral proteomes requires the use of specific prediction tools. PMID:28250370

  4. Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance1[OPEN

    PubMed Central

    Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee; Badger, John

    2017-01-01

    The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery. PMID:27879387

  5. Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties

    PubMed Central

    Aronsson, Christopher; Dånmark, Staffan; Zhou, Feng; Öberg, Per; Enander, Karin; Su, Haibin; Aili, Daniel

    2015-01-01

    Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials. PMID:26370878

  6. The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.

    PubMed

    Hohl, Marcel; Kwon, Youngho; Galván, Sandra Muñoz; Xue, Xiaoyu; Tous, Cristina; Aguilera, Andrés; Sung, Patrick; Petrini, John H J

    2011-09-04

    The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.

  7. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    2012-03-26

    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  8. Electrostatic contribution to the thermodynamic and kinetic stability of the homotrimeric coiled coil Lpp-56: A computational study.

    PubMed

    Bjelić, Sasa; Wieninger, Silke; Jelesarov, Ilian; Karshikoff, Andrey

    2008-02-15

    The protein moiety of the Braun's E. coli outer membrane lipoprotein (Lpp-56) is an attractive object of biophysical investigation in several aspects. It is a homotrimeric, parallel coiled coil, a class of coiled coils whose stability and folding have been studied only occasionally. Lpp-56 possesses unique structural properties and exhibits extremely low rates of folding and unfolding. It is natural to ask how the specificity of the structure determines the extraordinary physical chemical properties of this protein. Recently, a seemingly controversial data on the stability and unfolding rate of Lpp-56 have been published (Dragan et al., Biochemistry 2004;43: 14891-14900; Bjelic et al., Biochemistry 2006;45:8931-8939). The unfolding rate constant measured using GdmCl as the denaturing agent, though extremely low, was substantially higher than that obtained on the basis of thermal unfolding. If this large difference arises from the effect of screening of electrostatic interactions induced by GdmCl, electrostatic interactions would appear to be an important factor determining the unusual properties of Lpp-56. We present here a computational analysis of the electrostatic properties of Lpp-56 combining molecular dynamics simulations and continuum pK calculations. The pH-dependence of the unfolding free energy is predicted in good agreement with the experimental data: the change in DeltaG between pH 3 and pH 7 is approximately 60 kJ mol(-1). The results suggest that the difference in the stability of the protein observed using different experimental methods is mainly because of the effect of the reduction of electrostatic interactions when the salt (GdmCl) concentration increases. We also find that the occupancy of the interhelical salt bridges is unusually high. We hypothesize that electrostatic interactions, and the interhelical salt bridges in particular, are an important factor determining the low unfolding rate of Lpp-56.

  9. Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier

    PubMed Central

    Miller, Keith D.; Roque, Richard; Clegg, Christopher H.

    2014-01-01

    Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain. PMID:25494044

  10. Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

    PubMed Central

    Callahan, Kathryn E.; Hickman, Alison B.; Jones, Charles E.; Ghirlando, Rodolfo; Furano, Anthony V.

    2012-01-01

    The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival. PMID:21937507

  11. The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

    PubMed Central

    Greco, A; Mariani, C; Miranda, C; Lupas, A; Pagliardini, S; Pomati, M; Pierotti, M A

    1995-01-01

    Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences. PMID:7565764

  12. X-Ray Crystal Structure of a TRPM Assembly Domain Reveals An Antiparallel Four-Stranded Coiled-Coil

    SciTech Connect

    Fujiwara, Y.; Minor, D.L.; Jr.

    2009-05-18

    Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.

  13. The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

    PubMed

    Santos-Valle, Patricia; Guijarro-Muñoz, Irene; Cuesta, Angel M; Alonso-Camino, Vanesa; Villate, Maider; Alvarez-Cienfuegos, Ana; Blanco, Francisco J; Sanz, Laura; Alvarez-Vallina, Luis

    2012-01-01

    Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

  14. The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

    PubMed Central

    Santos-Valle, Patricia; Guijarro-Muñoz, Irene; Cuesta, Ángel M.; Alonso-Camino, Vanesa; Villate, Maider; Álvarez-Cienfuegos, Ana; Blanco, Francisco J.; Sanz, Laura; Álvarez-Vallina, Luis

    2012-01-01

    Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling. PMID:22723936

  15. The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

    PubMed

    Soeda, Shou; Yamada-Nomoto, Kaori; Ohsugi, Miho

    2016-10-01

    Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.

  16. Coiled-coil forming peptides for the induction of silver nanoparticles.

    PubMed

    Božič Abram, Sabina; Aupič, Jana; Dražić, Goran; Gradišar, Helena; Jerala, Roman

    2016-04-08

    Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV-VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings.

  17. Membrane interactions of fusogenic coiled-coil peptides: implications for lipopeptide mediated vesicle fusion.

    PubMed

    Rabe, Martin; Schwieger, Christian; Zope, Harshal R; Versluis, Frank; Kros, Alexander

    2014-07-08

    Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of molecules. However, little is known about the actual fusion mechanism at the molecular level. In this study we examine a system that models the key features of this process. For the molecular recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called 'E' (EIAALEK)3 and 'K' (KIAALKE)3 were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides 'E', 'K', and their parallel coiled-coil complex 'E/K' with the phospholipid membranes and thereby mimicked the pre- and postfusion states, respectively. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramolecular chemistry and biomedicine.

  18. Rho-associated coiled-coil containing kinases (ROCK): structure, regulation, and functions.

    PubMed

    Julian, Linda; Olson, Michael F

    2014-01-01

    Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase. (1)(-) (5) They belong to the AGC family of serine/threonine kinases (6) and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK.

  19. Thermodynamic aspects in a simplified model for the folding of two-stranded coiled-coils

    NASA Astrophysics Data System (ADS)

    Prolongo, Silvia G.; Rubio, Ana M.; Rey, Antonio

    2000-12-01

    We have investigated the thermodynamic properties of a simple model representing the thermal folding/unfolding transition of two-stranded coiled-coils. The transition temperature and the energy change for the process are analyzed in terms of the peptide concentration, using the standard properties and calculations involved in experimental work. The integration of the heat capacity curves provides realistic and correct results for the model, as it does the variation of the transition temperature with concentration. On the other hand, the van't Hoff analysis of the equilibrium constant for the unfolding process produces apparently odd results. They can only be rationalized through a careful analysis of the reaction stoichiometry, according to the reference state defined for the very simple model interactions, and the definition of the unfolded state. This point is extensively discussed, for its possible implications in the correct analysis of this and other simulation models.

  20. Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies.

    PubMed

    Sharma, Alok K; Sawhney, Paramvir; Memisoglu, Gonen; Rigby, Alan C

    2012-01-01

    Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.

  1. Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization.

    PubMed

    Morii, H; Takenawa, T; Arisaka, F; Shimizu, T

    1997-02-18

    The kinesin heavy chain consists of an N-terminal globular domain, referred to as the motor domain, a rod-like middle region, and a C-terminal domain. In this study, the human kinesin neck region, the region adjacent to the motor domain which promotes dimerization, has been investigated. First, we predicted coiled-coil regions including the neck region by our newly devised statistical method. The sequence (335-372) was predominated by a unique heptad amphipathy. A comparison of the bacterially expressed human kinesin heavy chain fragments, K349 (1-349), a monomeric motor domain, and K379 (1-379), a dimer, by circular dichroism (CD) spectroscopy showed that K379 had more alpha-helical content. Chemically synthesized peptides, (332-349), (350-379), and (332-369), gave CD spectra with an alpha-helix-rich pattern, but the spectra varied depending on the peptide concentration. Analysis of the molar ellipticity at 222 nm indicated that those peptides were in monomer-dimer equilibria, and the dissociation isotherms established dissociation constants of 9.6 mM. 60 microM, and 62 nM for the above peptides, respectively. Sedimentation equilibrium measurements verified that the peptide (332-369) existed as a dimeric form. These results strongly suggest that the sequence from 332 to 369 of the neck region forms an alpha-helical coiled coil. The differential peptide of K349 and K379, (350-379), did not show sufficient ability to make K379 dimeric. It is likely that the region (350-379) forms a stable alpha-helical coiled coil only together with the (332-349) region. Fluorescence energy transfer studies of [Cys363]-(332-369) labeled with a fluorescence donor and an acceptor revealed that the peptide formed a parallel coiled coil. This coiled coil was thermodynamically stable against urea and thermal denaturation, and peptide exchange of the coiled coil was undetectable, or extremely slow, at neutral pH. The dissociation free energy was estimated to be 57.7 kJ mol-1 at a peptide

  2. Differential backbone dynamics of companion helices in the extended helical coiled-coil domain of a bacterial chemoreceptor

    PubMed Central

    Bartelli, Nicholas L; Hazelbauer, Gerald L

    2015-01-01

    Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four-helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent-exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin-labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane-distal tight turn (N-helix), and the other carboxyl terminal (C-helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide-spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N- and C-helices. For bilayer-inserted receptors, EPR spectra from sites in the membrane-distal protein-interaction region and throughout the C-helix were typical of well-structured helices. In contrast, for approximately two-thirds of the N-helix, from its origin as the AS-2 helix of the membrane-proximal HAMP domain to the beginning of the membrane-distal protein-interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four-helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function. PMID:26257396

  3. An engineered coiled-coil polypeptide assembled onto quantum dots for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hao; Yang, Jie; Song, Ji-Tao; Zhang, Lin; Fang, Bi-Yun; Zhao, Dong-Hui; Xia, Rui-Xue; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2015-12-01

    Quantum dot (QD)-polypeptide probes have been developed through the specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and CdSe/ZnS core-shell QDs. The size and charge of a QD-polypeptide can be tuned by using different coiled-coil polypeptides. Compared to glutathione-capped QDs (QD-GSH), QD-polypeptide probes showed an approximately two- to three-fold luminescence increase, and the luminescence increase was not obviously related to the charge of the polypeptide. QD-polypeptide probes with different charge have a great effect on nonspecific cellular uptake. QD-polypeptide probes with negative charge exhibited lower nonspecific cellular uptake in comparison to the QD-GSH, while positively charged QD-polypeptide probes presented higher cellular uptake than the QD-GSH. A targeted QD-ARGD probe can obviously increase targeted cellular uptake in α v β 3 overexpressing HeLa cells compared to QD-A. In addition, QD-polypeptide probes showed lower in vitro cytotoxicity compared to the original QDs. These results demonstrate that these QD-polypeptide probes with high specific cellular uptake, high fluorescence intensity and low background noise are expected to have great potential applications in targeted cell imaging.

  4. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    PubMed Central

    Lesne, E.; Krammer, E.-M.; Dupre, E.; Locht, C.; Lensink, M. F.

    2016-01-01

    ABSTRACT The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. PMID:26933056

  5. Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance.

    PubMed Central

    Lauzon, A M; Fagnant, P M; Warshaw, D M; Trybus, K M

    2001-01-01

    Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance. PMID:11259302

  6. Structural Comparisons of Apo- and Metalated Three-Stranded Coiled Coils Clarify Metal Binding Determinants in Thiolate Containing Designed Peptides

    SciTech Connect

    Chakraborty, Saumen; Touw, Debra S.; Peacock, Anna F.A.; Stuckey, Jeanne; Pecoraro, Vincent L.

    2010-11-05

    Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metalated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metalated or nonmetalated forms and no examples where an apo structure and the corresponding metalated peptide assembly have both been defined by X-ray crystallography. Herein we present X-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a {yields} g)) CSL9C (CS = Coil Ser) and CSL19C in their nonmetalated forms, determined to 1.36 and 2.15 {angstrom} resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II), and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C){sub 3} show alternate conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C){sub 3} are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming X-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These 'apo' structures are used to clarify the changes in metal site organization between metalated As(CSL9C){sub 3} and to speculate on the differential basis of Hg

  7. Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A.

    PubMed

    Ren, Jinqi; Huo, Lin; Wang, Wenjuan; Zhang, Yong; Li, Wei; Lou, Jizhong; Xu, Tao; Feng, Wei

    2016-02-12

    Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3.

  8. Structural Correlation of the Neck Coil with the Coiled-coil (CC1)-Forkhead-associated (FHA) Tandem for Active Kinesin-3 KIF13A*

    PubMed Central

    Ren, Jinqi; Huo, Lin; Wang, Wenjuan; Zhang, Yong; Li, Wei; Lou, Jizhong; Xu, Tao; Feng, Wei

    2016-01-01

    Processive kinesin motors often contain a coiled-coil neck that controls the directionality and processivity. However, the neck coil (NC) of kinesin-3 is too short to form a stable coiled-coil dimer. Here, we found that the coiled-coil (CC1)-forkhead-associated (FHA) tandem (that is connected to NC by Pro-390) of kinesin-3 KIF13A assembles as an extended dimer. With the removal of Pro-390, the NC-CC1 tandem of KIF13A unexpectedly forms a continuous coiled-coil dimer that can be well aligned into the CC1-FHA dimer. The reverse introduction of Pro-390 breaks the NC-CC1 coiled-coil dimer but provides the intrinsic flexibility to couple NC with the CC1-FHA tandem. Mutations of either NC, CC1, or the FHA domain all significantly impaired the motor activity. Thus, the three elements within the NC-CC1-FHA tandem of KIF13A are structurally interrelated to form a stable dimer for activating the motor. This work also provides the first direct structural evidence to support the formation of a coiled-coil neck by the short characteristic neck domain of kinesin-3. PMID:26680000

  9. Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers.

    PubMed

    Weinert, Christopher; Morger, Damien; Djekic, Aleksandra; Grütter, Markus G; Mittl, Peer R E

    2015-06-04

    Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1β in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

  10. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    PubMed Central

    Woo, Sun Young; Lee, Hwankyu

    2016-01-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect. PMID:26926570

  11. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics.

    PubMed

    Woo, Sun Young; Lee, Hwankyu

    2016-03-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

  12. All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

    NASA Astrophysics Data System (ADS)

    Woo, Sun Young; Lee, Hwankyu

    2016-03-01

    Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

  13. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression.

    PubMed

    Wiedemann, Tobias; Hofbaur, Stefan; Loell, Eva; Rieder, Gabriele

    2016-09-29

    Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions.

  14. A C-Terminal Coiled-Coil Region of CagL is Responsible for Helicobacter Pylori-Induced Il-8 Expression

    PubMed Central

    Wiedemann, Tobias; Hofbaur, Stefan; Loell, Eva; Rieder, Gabriele

    2016-01-01

    Interleukin-8 (IL-8) is a potent neutrophil-activating chemokine which triggers the infiltration and migration of neutrophils into areas of bacterial infection. Helicobacter pylori-infected patient studies as well as animal models have revealed that H. pylori type I strains carrying an intact cytotoxin-associated gene pathogenicity island (cag-PAI) with a functional type IV secretion system (T4SS) induce IL-8 expression and secretion in gastric mucosa. This gastric mucosal IL-8 expression correlates with severe histological changes due to H. pylori infection. In the present study, we explored a new recognition pattern on the bacterial adhesion protein CagL inducing IL-8 expression in H. pylori-infected host cells. To analyze the secreted IL-8 concentration, we performed IL-8 enzyme-linked immunosorbent assay (ELISA). To investigate the H. pylori-induced IL-8 expression on the transcriptional level, we transiently transfected gastric epithelial cells (AGS) with a human IL-8 luciferase reporter construct. The results of this study demonstrate that specifically the C-terminal coiled-coil region of the H. pylori CagL protein, a protein described to be located on the tip of the T4SS-pilus, is responsible for several in vitro observations: 1) H. pylori-induced IL-8 secretion via the transforming growth factor (TGF)-α activated epidermal growth factor-receptor (EGF-R) signaling pathway; 2) H. pylori-induced elongation of the cells, a typical CagA-induced phenotype; and 3) the bridging of the T4SS to its human target cells. This novel bacterial-host recognition sequence allows a new insight into how H. pylori induces the inflammatory response in gastric epithelial cells and facilitates the development of precancerous conditions. PMID:27766167

  15. Structural Dynamics of the Vimentin Coiled-coil Contact Regions Involved in Filament Assembly as Revealed by Hydrogen-Deuterium Exchange.

    PubMed

    Premchandar, Aiswarya; Mücke, Norbert; Poznański, Jarosław; Wedig, Tatjana; Kaus-Drobek, Magdalena; Herrmann, Harald; Dadlez, Michał

    2016-11-25

    Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and to investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of the α-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within the full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, hydrogen-deuterium exchange analysis of the point-mutated variants directly demonstrated the active role of the IF consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer/tetramer interaction via "cross-coiling" as the first step of the assembly process.

  16. Klotho gene delivery ameliorates renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats by suppressing the Rho-associated coiled-coil kinase signaling pathway.

    PubMed

    Deng, Minghong; Luo, Yumei; Li, Yunkui; Yang, Qiuchen; Deng, Xiaoqin; Wu, Ping; Ma, Houxun

    2015-07-01

    The present study aimed to investigate whether klotho gene delivery attenuated renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats. A recombinant adeno-associated virus (rAAV) carrying mouse klotho full-length cDNA (rAAV.mKL), was constructed for in vivo investigation of klotho expression. Diabetes was induced in rats by a single tail vein injection of 60 mg/kg streptozotocin. Subsequently, the diabetic rats received an intravenous injection of rAAV.mKL, rAAV.green fluorescent protein (GFP) or phosphate-buffered saline (PBS). The Sprague-Dawley rat group received PBS and served as the control group. After 12 weeks, all the rats were sacrificed and ELISA, immunohistochemical and histological analyses, fluorescence microscopy, semi-quantitative reverse transcription-polymerase chain reaction and western blottin were performed. A single dose of rAAV.mKL was found to prevent the progression of renal hypertrophy and fibrosis for at least 12 weeks (duration of study). Klotho expression was suppressed in the diabetic rats, but was increased by rAAV.mKL delivery. rAAV.mKL significantly suppressed diabetes-induced renal hypertrophy and histopathological changes, reduced renal collagen fiber generation and decreased kidney hypertrophy index. In addition, rAAV.mKL decreased the protein expression levels of fibronectin and vimentin, while it downregulated the mRNA expression and activity of Rho-associated coiled-coil kinase (ROCK)I in the kidneys of the diabetic rats. These results indicated that klotho gene delivery ameliorated renal hypertrophy and fibrosis in diabetic rats, possibly by suppressing the ROCK signaling pathway. This may offer a novel approach for the long-term control and renoprotection of diabetes.

  17. Hydration dynamics at fluorinated protein surfaces.

    PubMed

    Kwon, Oh-Hoon; Yoo, Tae Hyeon; Othon, Christina M; Van Deventer, James A; Tirrell, David A; Zewail, Ahmed H

    2010-10-05

    Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface.

  18. Hydration dynamics at fluorinated protein surfaces

    PubMed Central

    Kwon, Oh-Hoon; Yoo, Tae Hyeon; Othon, Christina M.; Van Deventer, James A.; Tirrell, David A.; Zewail, Ahmed H.

    2010-01-01

    Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface. PMID:20855583

  19. The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains.

    PubMed

    Agou, Fabrice; Traincard, François; Vinolo, Emilie; Courtois, Gilles; Yamaoka, Shoji; Israël, Alain; Véron, Michel

    2004-07-02

    NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.

  20. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  1. Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer.

    PubMed

    Pluhackova, Kristyna; Wassenaar, Tsjerk A; Kirsch, Sonja; Böckmann, Rainer A

    2015-03-26

    A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

  2. Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5.

    PubMed

    Li, Chuanzhao; Plugariu, Carmela G; Bajgier, Joanna; White, John R; Liefer, Kristin M; Wu, Sheng-Jiun; Chaiken, Irwin

    2002-01-01

    Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.

  3. RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains.

    PubMed

    Ben-Ami, Hagit Cohen; Yassin, Lina; Farah, Hanna; Michaeli, Avner; Eshel, Margalit; Treinin, Millet

    2005-07-29

    Members of the RIC-3 gene family are effectors of nicotinic acetylcholine receptor (nAChR) expression in vertebrates and invertebrates. In Caenorhabditis elegans RIC-3 is needed for functional expression of multiple nAChRs, including the DEG-3/DES-2 nAChR. Effects of RIC-3 on DEG-3/DES-2 functional expression are found in vivo and following heterologous expression in Xenopus leavis oocytes. We now show that in X. leavis oocytes RIC-3 also affects the kinetics and agonist affinity properties of the DEG-3/DES-2 receptor. Because these effects are mimicked by increasing the ratio of DEG-3 subunits within DEG-3/DES-2 receptors, this suggests that RIC-3 may preferentially promote maturation of DEG-3-rich receptors. Indeed, effects of RIC-3 on functional expression of DEG-3/DES-2 positively correlate with the DEG-3 to DES-2 ratio. All RIC-3 family members have two transmembrane domains followed by one or two coiled-coil domains. Here we show that the effects of RIC-3 on functional expression and on receptor properties are mediated by the transmembrane domains and do not require the coiled-coil domains. In agreement with this, mammals express a RIC-3 transcript lacking the coiled-coil domain that is capable of promoting DEG-3/DES-2 functional expression. Last, we show that RIC-3 affects DEG-3 quantity, suggesting stabilization of receptors or receptor intermediates by RIC-3. Together our results suggest that subunit-specific interactions of RIC-3 with nAChR subunits, mediated by the transmembrane domains, are sufficient for the effects of RIC-3 on nAChR quantity and quality.

  4. Ursolic Acid Suppresses Hepatitis B Virus X Protein-mediated Autophagy and Chemotherapeutic Drug Resistance.

    PubMed

    Chang, Ching-Dong; Lin, Ping-Yuan; Hsu, Jue-Liang; Shih, Wen-Ling

    2016-10-01

    Hepatitis B virus X (HBx) protein is a multifunctional oncoprotein that affects diverse cell activities via regulation of various host cell signaling pathways. The current investigation demonstrated that ursolic acid (UA), a pentacyclic triterpenoid, protected hepatoma cells and reduced HBx-mediated autophagy through modulation of Ras homolog gene family member A (RhoA). Low-level ectopic HBx expression in Huh7 cells induced more significant autophagosome formation than high-level HBx expression. HBx activated beclin-1 promoter and enhanced the beclin-1 protein expression under low HBx expression. Transcription factor AP-1 played an essential function in HBx-mediated beclin-1 promoter activation. Inhibition of RhoA and its downstream effector Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) alleviated HBx-mediated autophagy significantly. Transiently-expressed HBx elicited an increased RhoA-GTP level, as well as phospho-ROCK1 transient accumulation. Utilization of transactivation-deficient HBx demonstrated that the transactivation activity of HBx is required for autophagy induction. Furthermore, UA suppressed HBx-mediated RhoA activation, beclin-1 promoter activation and subsequent autophagy induction, while, most importantly, reversed HBx-induced anti-cancer drug resistance.

  5. Two Complementary Approaches for the Controlled Release of Biomolecules Immobilized via Coiled-Coil Interactions: Peptide Core Mutations and Multivalent Presentation.

    PubMed

    Murschel, Frederic; Fortier, Charles; Jolicoeur, Mario; Hodges, Robert S; De Crescenzo, Gregory

    2017-03-13

    We have developed a heterodimeric coiled-coil system based on two complementary peptides, namely (EVSALEK)5 and (KVSALKE)5, or E and K, for the attachment of E-tagged biomolecules onto K-decorated biomaterials. We here explore two approaches to control the strength and the stability of the E/K coiled-coil complex, and thus its potential for the controlled release of biomolecules. Those are Leucine-to-Alanine mutations in the K peptide (4 peptides with 0 to 3 mutations) and multivalent presentation of the E peptide (6 bio-objects from monomeric to dimeric and n-meric). Using E-tagged growth factors and nanoparticles as models, SPR-based assays performed under continuous flow indicated that the release rate was strongly affected by both approaches independently, and that the strength of the capture could be finely tuned over a wide range (apparent dissociation constant from 0.12 pM to 270 nM). Further release assays carried out in well-plates showed that the multivalent presentation only had a significant influence in this setup since the wells were not rinsed under continuous flow.

  6. An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the -Helical Trimeric Coiled Coil

    SciTech Connect

    Bhardwaj, A.; Walker-Kopp, N; Casjens, S; Cingolani, G

    2009-01-01

    Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated {alpha}-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 {angstrom}. gp26 tail needles display a high level of structural stability in solution, with Tm (temperature of melting) between 85 and 95 C. To determine how the structural stability of these phage fibers correlates with the length of the {alpha}-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the {alpha}-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the {alpha}-helical core.

  7. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  8. Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation.

    PubMed

    Röhrig, Horst; Schmidt, Jürgen; Colby, Thomas; Bräutigam, Anne; Hufnagel, Peter; Bartels, Dorothea

    2006-08-01

    Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.

  9. Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

    PubMed Central

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith

    2011-01-01

    Abstract Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy. PMID:21453126

  10. Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18.

    PubMed

    d'Avignon, D André; Bretthorst, G Larry; Holtzer, Marilyn Emerson; Schwarz, Kathleen A; Angeletti, Ruth Hogue; Mints, Lisa; Holtzer, Alfred

    2006-10-15

    The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate

  11. [First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains].

    PubMed

    Kapelinskaia, T V; Kagramanova, A S; Korolev, A L; Mukha, D V

    2011-02-01

    The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.

  12. A heterozygous dominant-negative mutation in the coiled-coil domain of STAT1 is the cause of autosomal-dominant Mendelian susceptibility to mycobacterial diseases.

    PubMed

    Ueki, Masahiro; Yamada, Masafumi; Ito, Kenta; Tozawa, Yusuke; Morino, Saeko; Horikoshi, Yuho; Takada, Hidetoshi; Abdrabou, Shimaa Said Mohamed Ali; Takezaki, Shunichiro; Kobayashi, Ichiro; Ariga, Tadashi

    2017-01-01

    Heterozygous dominant-negative mutations of STAT1 are responsible for autosomal-dominant Mendelian susceptibility to mycobacterial diseases (AD-MSMD). So far, only 7 mutations have been previously described and are localized to 3 domains: the DNA-binding domain, the SH2 domain, and the tail segment. In this study, we demonstrated the first coiled-coil domain (CCD) mutation of c.749G>C, p.G250A (G250A) in STAT1 as a genetic cause of AD-MSMD in a patient with mycobacterial multiple osteomyelitis. This de novo heterozygous mutation was shown to have a dominant-negative effect on the gamma-activated sequence (GAS) transcriptional activity following IFN-γ stimulation, which could be attributable to the abolished phosphorylation of STAT1 from the wild-type (WT) allele. The three-dimensional structure of STAT1 revealed the G250 residue was located distant from a cluster of residues affected by gain-of-function mutations responsible for chronic mucocutaneous candidiasis.

  13. Overexpression of Transforming Acidic Coiled Coil‑Containing Protein 3 Reflects Malignant Characteristics and Poor Prognosis of Glioma

    PubMed Central

    Sun, Ying; Tian, Yu; Wang, Guang-Zhi; Zhao, Shi-Hong; Han, Bo; Li, Yong-Li; Jiang, Chuan-Lu

    2017-01-01

    Gliomas are malignant primary brain tumors with poor prognosis. Recently, research was indicative of a tight connection between tumor malignancy and genetic alterations. Here, we propose an oncogenic implication of transforming acidic coiled-coil-containing protein 3 (TACC3) in gliomas. By comprehensively analyzing the Chinese glioma genome atlas (CGGA) and publicly available data, we demonstrated that TACC3 were overexpressed along with glioma grade and served as an independent negative prognostic biomarker for glioma patients. Functions’ annotations and gene sets’ enrichment analysis suggested that TACC3 may participate in cell cycle, DNA repair, epithelium-mesenchymal transition and other tumor-related biological processes and molecular pathways. Patients with high TACC3 expression showed CD133+ stem cell properties, glioma plasticity and shorter overall survival time under chemo-/radio-therapy. Additionally, a TACC3 associated the miRNA-mRNA network was constructed based on in silico prediction and expression pattern, which provide a foundation for further detection of TACC3-miRNA-mRNA axis function. Collectively, our observations identify TACC3 as an oncogene of tumor malignancy, as well as a prognostic and motoring biomarker for glioma patients. PMID:28273854

  14. Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics.

    PubMed

    Nelson, Josh D; Kinkead, Heather; Brunel, Florence M; Leaman, Dan; Jensen, Richard; Louis, John M; Maruyama, Toshiaki; Bewley, Carole A; Bowdish, Katherine; Clore, G Marius; Dawson, Philip E; Frederickson, Shana; Mage, Rose G; Richman, Douglas D; Burton, Dennis R; Zwick, Michael B

    2008-07-20

    Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35(CCG)-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

  15. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  16. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    NASA Astrophysics Data System (ADS)

    Fat'yanov, O. V.; Asimow, P. D.

    2015-10-01

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  17. Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

    SciTech Connect

    Fat’yanov, O. V. Asimow, P. D.

    2015-10-15

    We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documented methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen

  18. cDNA cloning, localization, and candidate binding partners of acid-extractable vitelline-coat protein Ci-v-Themis-like in the ascidian Ciona intestinalis.

    PubMed

    Otsuka, Kei; Yamada, Lixy; Sawada, Hitoshi

    2013-10-01

    Ascidians are hermaphrodites, although several ascidian species show self-sterility because of the occurrence of a self/nonself-recognition system called the self-incompatibility system. We previously reported that two pairs of sperm polycystin 1-like receptors, s-Themis-A and s-Themis-B, and egg fibrinogen-like ligands, v-Themis-A and v-Themis-B, are responsible for self-incompatibility in the ascidian Ciona intestinalis. Our previous results showed that v-Themis-A and v-Themis-B were hardly extracted from the vitelline coat (VC) by acid treatment, which is not in accordance with a report that an acid-extractable VC factor has the ability to distinguish self- from nonself-sperm. These results led us to explore a novel factor from acid-extractable VC proteins that could be involved in self-incompatibility. Here, we report cDNA cloning, expression, and localization of Ci-v-Themis-like, a major acid-extractable VC protein. This protein has a fibrinogen-like domain, as do v-Themis-A and v-Themis-B, but it showed no polymorphisms. Phylogenic analysis suggested that Ci-v-Themis-like is an ancestral protein of v-Themis-A and v-Themis-B. Whole mount in situ hybridization revealed that Ci-v-Themis-like mRNA is expressed in the ovary and testis. Western blotting and immunocytochemistry showed the occurrence of Ci-v-Themis-like in developing oocytes and on the VC of mature eggs. Yeast two-hybrid screenings using testis and ovary libraries revealed candidate interacting proteins; among these candidates, we succeeded in identifying several testis-specific proteins, including sperm proteases and coiled-coil-domain-containing proteins. The results suggest that Ci-v-Themis-like and its binding partners are involved in sperm binding to the VC prior to the allorecognition process during C. intestinalis fertilization.

  19. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    PubMed

    Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G Marius; Wlodawer, Alexander

    2013-01-01

    A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

  20. N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA2 subunit to form an N cap that terminates the triple-stranded coiled coil

    PubMed Central

    Chen, Jue; Skehel, John J.; Wiley, Don C.

    1999-01-01

    The structure of a stable recombinant ectodomain of influenza hemagglutinin HA2 subunit, EHA2 (23–185), defined by proteolysis studies of the intact bacterial-expressed ectodomain, was determined to 1.9-Å resolution by using x-ray crystallography. The structure reveals a domain composed of N- and C-terminal residues that form an N cap terminating both the N-terminal α-helix and the central coiled coil. The N cap is formed by a conserved sequence, and part of it is found in the neutral pH conformation of HA. The C-terminal 23 residues of the ectodomain form a 72-Å long nonhelical structure ordered to within 7 residues of the transmembrane anchor. The structure implies that continuous α helices are not required for membrane fusion at either the N or C termini. The difference in stability between recombinant molecules with and without the N cap sequences suggests that additional free energy for membrane fusion may become available after the formation of the central triple-stranded coiled coil and insertion of the fusion peptide into the target membrane. PMID:10430879

  1. Dimerization of TRAF-interacting protein (TRAIP) regulates the mitotic progression.

    PubMed

    Park, I Seul; Jo, Ku-Sung; Won, Hyung-Sik; Kim, Hongtae

    2015-08-07

    The homo- or hetero-dimerization of proteins plays critical roles in the mitotic progression. The TRAF-interacting protein (TRAIP) is crucial in early mitotic progression and chromosome alignment defects in the metaphase. The TRAIP is a 469 amino acid protein, including the Really Interesting New Gene (RING), coiled-coil (CC), and leucine zipper (LZ) domain. In general, the CC or LZ domain containing proteins forms homo- or hetero-dimerization to achieve its activity. In this study, a number of TRAIP mutants were used to define the TRAIP molecular domains responsible for its homo-dimerization. A co-immunoprecipitation assay indicated that the TRAIP forms homo-dimerization through the CC domain. The cells, expressing the CC domain-deleted mutant that could not form a homo-dimer, increased the mitotic index and promoted mitotic progression.

  2. Structural Proteins from Whelk Egg Capsule with Long Range Elasticity Associated with a Solid-state Phase Transition

    PubMed Central

    Wasko, S. Scott; Tay, Gavin; Schwaighofer, Andreas; Nowak, Christoph; Waite, J. Herbert; Miserez, Ali

    2014-01-01

    The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helix to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle x-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules. PMID:24350603

  3. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

    PubMed

    Veena, Mariswamy; Melvin, Prasad; Prabhu, Sreedhara Ashok; Shailasree, Sekhar; Shetty, Hunthrike Shekar; Kini, Kukkundoor Ramachandra

    2016-03-01

    Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction.

  4. Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats

    PubMed Central

    Manjula, Belur N.; Mische, Sheenah M.; Fischetti, Vincent A.

    1983-01-01

    Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of α-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats. PMID:16593365

  5. Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae

    PubMed Central

    Kawakita, Yoshito; Kinoshita, Miki; Furukawa, Yukio; Tulum, Isil; Tahara, Yuhei O.; Katayama, Eisaku

    2016-01-01

    ABSTRACT Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae. This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma “gliding motility” is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is

  6. Protein and amino acid nutrition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

  7. Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction.

    PubMed

    Inoue, Haruhiko; Hayashi, Nagao; Matsushita, Akane; Xinqiong, Liu; Nakayama, Akira; Sugano, Shoji; Jiang, Chang-Jie; Takatsuji, Hiroshi

    2013-06-04

    Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar "Modan." Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1-mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1-mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression (Pb1-ox) was also compromised in WRKY45 knockdown/Pb1-ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1-ox than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.

  8. Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 chain.

    PubMed

    Schéele, Susanne; Sasaki, Takako; Arnal-Estapé, Anna; Durbeej, Madeleine; Ekblom, Peter

    2006-07-01

    We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain.

  9. Development and characterization of recombinant human Fc:OX40L fusion protein linked via a coiled-coil trimerization domain.

    PubMed

    Morris, Nicholas P; Peters, Carmen; Montler, Ryan; Hu, Hong-Ming; Curti, Brendan D; Urba, Walter J; Weinberg, Andrew D

    2007-05-01

    OX40 (CD134) is a potent costimulatory molecule found on the surface of activated CD4(+) and CD8(+) T cells. Immunotherapy with OX40 agonists administered in vivo has demonstrated efficacy in several murine tumor models. A phase I clinical trial is currently underway in patients with advanced cancer using a mouse anti-CD134 monoclonal antibody. Therapy with this antibody will likely be limited to one cycle because patients develop neutralizing human anti-mouse antibody (HAMA). Therefore, we developed a humanized OX40 agonist that links the extracellular domain of human OX40L to the Fc domain of human IgG(1) via a trimerizing isoleucine zipper domain (ILZ). Physical characterization by velocity sedimentation revealed that this novel construct, hFcILZOX40L, was assembled into hexamers in which the Fc domains formed three disulfide-bonded dimers and the ILZ-OX40L domains formed two trimers. Trimerization of the ILZ domain was necessary to achieve appropriate assembly. In vitro biologic activity of the hFcILZOX40L hexamer was equivalent to the activity of agonist antibodies in plate-bound assays and was superior when the agonists were tested as soluble agents. Our ultimate goal is to use this recombinant molecule in a future clinical trial, and we feel that the OX40L hexamer will have equivalent or superior agonist activity in vivo when compared to an anti-OX40 antibody.

  10. Development and Characterization of Recombinant Human Fc:OX40L fusion protein linked via a coiled-coil trimerization domain

    PubMed Central

    Morris, Nicholas P.; Peters, Carmen; Montler, Ryan; Hu, Hong-Ming; Curti, Brendan D.; Urba, Walter J.; Weinberg, Andrew D.

    2007-01-01

    OX40 (CD134) is a potent costimulatory molecule found on the surface of activated CD4+ and CD8+ T cells. Immunotherapy with OX40 agonists administered in vivo has demonstrated efficacy in several murine tumor models. A phase I clinical trial is currently underway in patients with advanced cancer using a mouse anti-CD134 monoclonal antibody. Therapy with this antibody will likely be limited to one cycle because patients develop neutralizing human anti-mouse antibody (HAMA). Therefore, we developed a humanized OX40 agonist that links the extracellular domain of human OX40L to the Fc domain of human IgG1 via a trimerizing isoleucine zipper domain (ILZ). Physical characterization by velocity sedimentation revealed that this novel construct, hFcILZOX40L, was assembled into hexamers in which the Fc domains formed three disulfide-bonded dimers and the ILZ-OX40L domains formed two trimers. Trimerization of the ILZ domain was necessary to achieve appropriate assembly. In vitro biologic activity of the hFcILZOX40L hexamer was equivalent to the activity of agonist antibodies in plate-bound assays and was superior when the agonists were tested as soluble agents. Our ultimate goal is to use this recombinant molecule in a future clinical trial and we feel that the OX40L hexamer will have equivalent or superior agonist activity in vivo when compared to an anti-OX40 antibody. PMID:17374396

  11. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    PubMed

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

  12. Interaction of TACC proteins with the FHL family: implications for ERK signaling

    PubMed Central

    Lauffart, Brenda; Sondarva, Gautam V.; Gangisetty, Omkaram; Cincotta, Melissa

    2007-01-01

    The Transforming acidic coiled coil (TACC) proteins play a conserved role in normal development and tumorigenesis through interactions with multiple complexes involved in transcription, translation, and centrosomal dynamics. However, despite significant work on the function of TACC3 in the control of centrosomal mechanics, relatively little functional data is known about the family’s founding member, TACC1. From a continued analysis of clones isolated by an unbiased yeast two-hybrid assay, we now show direct physical interactions between the TACC1 and the FHL (Four and a Half LIM-only) family of proteins. The authenticity of these interactions was validated both in vitro and in cellular systems. The FHLs exhibit diverse biological roles such as the regulation of the actin cytoskeleton and are promiscuous coregulators for several transcription factors. The interaction of the endogenous TACC-FHL proteins is primarily localized to the nucleus. However, similar to FHL2, overexpression of TACC1A in HEK293 is able to sequester serum activated ERK to the cytoplasm. This has the effect of reducing the serum induced transcriptional response of the c-fos and c-jun genes. The observation that TACCs can interact with the FHLs and alter their serum induced activities raises the possibility that the TACCs participate in crosstalk between cell signaling pathways important for cancer development and tumor progression. The transforming acidic coiled coil genes are known to be important prognostic indicators for breast, ovarian and lung cancer. In this manuscript, we identify a novel interaction between the TACCs and the FHL protein family. This interaction has an affect on ERK and may in part explain the variable associations and changes in subcellular locations of each family with specific subtypes of malignancy. PMID:18481206

  13. Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins.

    PubMed Central

    Kastner, P; Perez, A; Lutz, Y; Rochette-Egly, C; Gaub, M P; Durand, B; Lanotte, M; Berger, R; Chambon, P

    1992-01-01

    Acute promyelocytic leukemia (APL) is due to a chromosomal t(15;17) translocation which involves a novel human gene, Myl, (also named PML) and the retinoic acid (RA) receptor alpha (RAR-alpha) gene. We report here the characterization of Myl and of the reciprocal MylRAR (PMLRAR) and RARMyl (RARPML) fusion transcripts which are found in two classes of APL patients. Myl displays similarities with a new family of proteins of which some members are fused to protooncogenes in the transforming proteins RFP-ret and T18. The speckled nuclear localization of Myl, as well as its sequence homology with the 52 kDa component of the RO/SSA ribonucleoprotein particle, suggest that Myl may be present in a ribonucleoprotein complex. In contrast to both Myl and RAR-alpha whose localization is essentially nuclear in the presence or absence of RA, MylRAR which is largely cytoplasmic in the absence of RA appears to be translocated to the nucleus in the presence of RA. Myl and MylRAR can associate in vitro and this association is mediated by a coiled coil in the Myl sequence. In vivo this association results in a colocalization of Myl and MylRAR which is identical to that of MylRAR alone. Studies of activation of transcription from the promoters of several RA target genes indicate that MylRARs have altered transcription activation properties when compared with RAR-alpha. Most notably, MylRAR represses markedly the activity of some RA target promoters in the absence of RA. Western blot analyses of patient samples show that MylRAR is expressed to a much higher level than wild type RAR-alpha originating from the normal allele. Taken together, these results suggest that MylRAR may interfere in a dominant manner with both Myl and RAR functions. Images PMID:1311253

  14. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

    PubMed Central

    Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

    2005-01-01

    We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

  15. A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis.

    PubMed

    Vicente-Manzanares, Miguel; Cabrero, José Román; Rey, Mercedes; Pérez-Martínez, Manuel; Ursa, Angeles; Itoh, Kazuyuki; Sánchez-Madrid, Francisco

    2002-01-01

    The possible involvement of the Rho-p160ROCK (Rho coiled-coil kinase) pathway in the signaling induced by the chemokine Stromal cell-derived factor (SDF)-1alpha has been studied in human PBL. SDF-1alpha induced activation of RhoA, but not that of Rac. RhoA activation was followed by p160ROCK activation mediated by RhoA, which led to myosin light chain (MLC) phosphorylation, which was dependent on RhoA and p160ROCK activities. The kinetics of MLC activation was similar to that of RhoA and p160ROCK. The role of this cascade in overall cell morphology and functional responses to the chemokine was examined employing different chemical inhibitors. Inhibition of either RhoA or p160ROCK did not block SDF-1alpha-induced short-term actin polymerization, but induced the formation of long spikes arising from the cell body, which were found to be microtubule based. This morphological change was associated with an increase in microtubule instability, which argues for an active microtubule polymerization in the formation of these spikes. Inhibition of the Rho-p160ROCK-MLC kinase signaling cascade at different steps blocked lymphocyte migration and the chemotaxis induced by SDF-1alpha. Our results indicate that the Rho-p160ROCK axis plays a pivotal role in the control of the cell shape as a step before lymphocyte migration toward a chemotactic gradient.

  16. Quantifying protein by bicinchoninic Acid.

    PubMed

    Simpson, Richard J

    2008-08-01

    INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu(+) generated under alkaline conditions at sites of complexes between Cu(2+) and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.

  17. The Rab interacting lysosomal protein (RILP) homology domain functions as a novel effector domain for small GTPase Rab36: Rab36 regulates retrograde melanosome transport in melanocytes.

    PubMed

    Matsui, Takahide; Ohbayashi, Norihiko; Fukuda, Mitsunori

    2012-08-17

    Small GTPase Rab functions as a molecular switch that drives membrane trafficking through specific interaction with its effector molecule. Thus, identification of its specific effector domain is crucial to revealing the molecular mechanism that underlies Rab-mediated membrane trafficking. Because of the large numbers of Rab isoforms in higher eukaryotes, however, the effector domains of most of the vertebrate- or mammalian-specific Rabs have yet to be determined. In this study we screened for effector molecules of Rab36, a previously uncharacterized Rab isoform that is largely conserved in vertebrates, and we succeeded in identifying nine Rab36-binding proteins, including RILP (Rab interacting lysosomal protein) family members. Sequence comparison revealed that five of nine Rab36-binding proteins, i.e. RILP, RILP-L1, RILP-L2, and JIP3/4, contain a conserved coiled-coil domain. We identified the coiled-coil domain as a RILP homology domain (RHD) and characterized it as a common Rab36-binding site. Site-directed mutagenesis of the RHD of RILP revealed the different contributions by amino acids in the RHD to binding activity toward Rab7 and Rab36. Expression of RILP in melanocytes, but not expression of its Rab36 binding-deficient mutants, induced perinuclear aggregation of melanosomes, and this effect was clearly attenuated by knockdown of endogenous Rab36 protein. Moreover, knockdown of Rab36 in Rab27A-deficient melanocytes, which normally exhibit perinuclear melanosome aggregation because of increased retrograde melanosome transport activity, caused dispersion of melanosomes from the perinucleus to the cell periphery, but knockdown of Rab7 did not. Our findings indicated that Rab36 mediates retrograde melanosome transport in melanocytes through interaction with RILP.

  18. A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

    PubMed Central

    Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A.; Favre, Gilles; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence. PMID:24092409

  19. A new protein-protein interaction sensor based on tripartite split-GFP association.

    PubMed

    Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-10-04

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

  20. Boehringer Mannheim award lecture 1995. La conference Boehringer Mannheim 1995. De novo design of alpha-helical proteins: basic research to medical applications.

    PubMed

    Hodges, R S

    1996-01-01

    The two-stranded alpha-helical coiled-coil is a universal dimerization domain used by nature in a diverse group of proteins. The simplicity of the coiled-coil structure makes it an ideal model system to use in understanding the fundamentals of protein folding and stability and in testing the principles of de novo design. The issues that must be addressed in the de novo design of coiled-coils for use in research and medical applications are (i) controlling parallel versus antiparallel orientation of the polypeptide chains, (ii) controlling the number of helical strands in the assembly (iii) maximizing stability of homodimers or heterodimers in the shortest possible chain length that may require the engineering of covalent constraints, and (iv) the ability to have selective heterodimerization without homodimerization, which requires a balancing of selectivity versus affinity of the dimerization strands. Examples of our initial inroads in using this de novo design motif in various applications include: heterodimer technology for the detection and purification of recombinant peptides and proteins; a universal dimerization domain for biosensors; a two-stage targeting and delivery system; and coiled-coils as templates for combinatorial helical libraries for basic research and drug discovery and as synthetic carrier molecules. The universality of this dimerization motif in nature suggests an endless number of possibilities for its use in de novo design, limited only by the creativity of peptide-protein engineers.

  1. Pex17p Is Required for Import of Both Peroxisome Membrane and Lumenal Proteins and Interacts with Pex19p and the Peroxisome Targeting Signal–Receptor Docking Complex in Pichia pastoris

    PubMed Central

    Snyder, William B.; Koller, Antonius; Choy, Aaron Jobu; Johnson, Monique A.; Cregg, James M.; Rangell, Linda; Keller, Gilbert A.; Subramani, Suresh

    1999-01-01

    Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Δ mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Δ mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal–receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs. PMID:10588639

  2. The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

    PubMed Central

    Dowling, L M; Crewther, W G; Inglis, A S

    1986-01-01

    Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming

  3. A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae

    PubMed Central

    1991-01-01

    The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion- deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze- substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature- sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments. PMID:2010462

  4. Structural characterization of Mumps virus fusion protein core

    SciTech Connect

    Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng . E-mail: Qiubs@sun.im.ac.cn; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn; Tien, Po . E-mail: tienpo@sun.im.ac.cn

    2006-09-29

    The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.

  5. A logical sequence search for S100B target proteins.

    PubMed Central

    McClintock, K. A.; Shaw, G. S.

    2000-01-01

    The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (alpha and beta), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of alpha and beta tubulin found at the alpha beta tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins. PMID:11106180

  6. Engineering the Dynamic Properties of Protein Networks through Sequence Variation

    PubMed Central

    2016-01-01

    The dynamic behavior of macromolecular networks dominates the mechanical properties of soft materials and influences biological processes at multiple length scales. In hydrogels prepared from self-assembling artificial proteins, stress relaxation and energy dissipation arise from the transient character of physical network junctions. Here we show that subtle changes in sequence can be used to program the relaxation behavior of end-linked networks of engineered coiled-coil proteins. Single-site substitutions in the coiled-coil domains caused shifts in relaxation time over 5 orders of magnitude as demonstrated by dynamic oscillatory shear rheometry and stress relaxation measurements. Networks with multiple relaxation time scales were also engineered. This work demonstrates how time-dependent mechanical responses of macromolecular materials can be encoded in genetic information. PMID:27924309

  7. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  8. Gravitropism and Lateral Root Emergence are Dependent on the Trans-Golgi Network Protein TNO1

    PubMed Central

    Roy, Rahul; Bassham, Diane C.

    2015-01-01

    The trans-Golgi network (TGN) is a dynamic organelle that functions as a relay station for receiving endocytosed cargo, directing secretory cargo, and trafficking to the vacuole. TGN-localized SYP41-interacting protein (TNO1) is a large, TGN-localized, coiled-coil protein that associates with the membrane fusion protein SYP41, a target SNARE, and is required for efficient protein trafficking to the vacuole. Here, we show that a tno1 mutant has auxin transport-related defects. Mutant roots have delayed lateral root emergence, decreased gravitropic bending of plant organs and increased sensitivity to the auxin analog 2,4-dichlorophenoxyacetic acid and the natural auxin 3-indoleacetic acid. Auxin asymmetry at the tips of elongating stage II lateral roots was reduced in the tno1 mutant, suggesting a role for TNO1 in cellular auxin transport during lateral root emergence. During gravistimulation, tno1 roots exhibited delayed auxin transport from the columella to the basal epidermal cells. Endocytosis to the TGN was unaffected in the mutant, indicating that bulk endocytic defects are not responsible for the observed phenotypes. Together these studies demonstrate a role for TNO1 in mediating auxin responses during root development and gravistimulation, potentially through trafficking of auxin transport proteins. PMID:26617617

  9. Acanthamoeba castellanii STAT Protein

    PubMed Central

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  10. The Postsynaptic Density Proteins Homer and Shank Form a Polymeric Network Structure

    SciTech Connect

    Hayashi, M.; Tang, C; Verpelli, C; Narayanan, R; Stearns, M; Xu, R; Li, H; Sala, C; Hayashi, Y

    2009-01-01

    The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

  11. Caenorhabditis elegans NONO-1: Insights into DBHS protein structure, architecture, and function.

    PubMed

    Knott, Gavin J; Lee, Mihwa; Passon, Daniel M; Fox, Archa H; Bond, Charles S

    2015-12-01

    Members of the Drosophila behavior/human splicing (DBHS) protein family have been characterized in the vertebrates Homo sapiens and Mus musculus, and the invertebrates Drosophila melanogaster and Chironomus tentans. Collectively, both vertebrate and invertebrate DBHS proteins function throughout gene regulation, largely but not always, within the nucleus. In this study, we report a structural and bioinformatic analysis of the DBHS protein family to guide future studies into DBHS protein function. To explore the structural plasticity of the family, we describe the 2.4 Å crystal structure of Caenorhabditis elegans non-POU domain-containing octamer-binding protein 1 (NONO-1). The structure is dimeric, with a domain arrangement consistent with mammalian DBHS proteins. Comparison with the DBHS structures available from H. sapiens reveals that there is inherent domain flexibility within the homologous DBHS region. Mapping amino acid similarity within the family to the NONO-1 dimer highlights the dimer interface, coiled-coil oligomerization motif, and putative RNA binding surfaces. Surprisingly, the interior surface of RNA recognition motif 2 (RRM2) that faces a large internal void is highly variable, but the external β2-β3 loops of RRM2 show remarkable preservation. Overall, the DBHS region is under strong purifying selection, whereas the sequences N- and C-terminal to the DBHS region are less constrained. The findings described in this study provide a molecular basis for further investigation into the mechanistic function of the DBHS protein family in biology.

  12. Dilatory is a Drosophila protein related to AZI1 (CEP131) that is located at the ciliary base and required for cilium formation

    PubMed Central

    Ma, Lina; Jarman, Andrew P.

    2011-01-01

    A significant number of ciliary disease genes have been found to encode proteins that localise to the basal body. By contrast, a large number of basal-body-associated proteins remain to be characterised. Here, we report the identification of a new basal body protein that is required for ciliogenesis in Drosophila. Dilatory (DILA) is a predicted coiled-coil protein homologous to vertebrate AZI1 (also known as CEP131). Mutations in dila specifically exhibit defects in ciliated cells (sensory neurons and sperm). Several features of the neuronal phenotype suggest a defect in intraflagellar transport. In sensory neuron cilia, DILA protein localises to the ciliary base, including the basal body and putative transition zone, and it interacts genetically with the ciliary coiled-coil protein, Uncoordinated. These data implicate DILA in regulating intraflagellar transport at the base of sensory cilia. PMID:21750193

  13. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  14. SLC27 fatty acid transport proteins.

    PubMed

    Anderson, Courtney M; Stahl, Andreas

    2013-01-01

    The uptake and metabolism of long chain fatty acids (LCFA) are critical to many physiological and cellular processes. Aberrant accumulation or depletion of LCFA underlie the pathology of numerous metabolic diseases. Protein-mediated transport of LCFA has been proposed as the major mode of LCFA uptake and activation. Several proteins have been identified to be involved in LCFA uptake. This review focuses on the SLC27 family of fatty acid transport proteins, also known as FATPs, with an emphasis on the gain- and loss-of-function animal models that elucidate the functions of FATPs in vivo and how these transport proteins play a role in physiological and pathological situations.

  15. Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

    PubMed Central

    McKnight, Nicole C; Jefferies, Harold B J; Alemu, Endalkachew A; Saunders, Rebecca E; Howell, Michael; Johansen, Terje; Tooze, Sharon A

    2012-01-01

    Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP–LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation. PMID:22354037

  16. Creation of Hybrid Nanorods From Sequences of Natural Trimeric Fibrous Proteins Using the Fibritin Trimerization Motif

    NASA Astrophysics Data System (ADS)

    Papanikolopoulou, Katerina; van Raaij, Mark J.; Mitraki, Anna

    Stable, artificial fibrous proteins that can be functionalized open new avenues in fields such as bionanomaterials design and fiber engineering. An important source of inspiration for the creation of such proteins are natural fibrous proteins such as collagen, elastin, insect silks, and fibers from phages and viruses. The fibrous parts of this last class of proteins usually adopt trimeric, β-stranded structural folds and are appended to globular, receptor-binding domains. It has been recently shown that the globular domains are essential for correct folding and trimerization and can be successfully substituted by a very small (27-amino acid) trimerization motif from phage T4 fibritin. The hybrid proteins are correctly folded nanorods that can withstand extreme conditions. When the fibrous part derives from the adenovirus fiber shaft, different tissue-targeting specificities can be engineered into the hybrid proteins, which therefore can be used as gene therapy vectors. The integration of such stable nanorods in devices is also a big challenge in the field of biomechanical design. The fibritin foldon domain is a versatile trimerization motif and can be combined with a variety of fibrous motifs, such as coiled-coil, collagenous, and triple β-stranded motifs, provided the appropriate linkers are used. The combination of different motifs within the same fibrous molecule to create stable rods with multiple functions can even be envisioned. We provide a comprehensive overview of the experimental procedures used for designing, creating, and characterizing hybrid fibrous nanorods using the fibritin trimerization motif.

  17. Why fibrous proteins are romantic.

    PubMed

    Cohen, C

    1998-01-01

    Here I give a personal account of the great history of fibrous protein structure. I describe how Astbury first recognized the essential simplicity of fibrous proteins and their paradigmatic role in protein structure. The poor diffraction patterns yielded by these proteins were then deciphered by Pauling, Crick, Ramachandran and others (in part by model building) to reveal alpha-helical coiled coils, beta-sheets, and the collagen triple helical coiled coil-all characterized by different local sequence periodicities. Longer-range sequence periodicities (or "magic numbers") present in diverse fibrous proteins, such as collagen, tropomyosin, paramyosin, myosin, and were then shown to account for the characteristic axial repeats observed in filaments of these proteins. More recently, analysis of fibrous protein structure has been extended in many cases to atomic resolution, and some systems, such as "leucine zippers," are providing a deeper understanding of protein design than similar studies of globular proteins. In the last sections, I provide some dramatic examples of fibrous protein dynamics. One example is the so-called "spring-loaded" mechanism for viral fusion by the hemagglutinin protein of influenza. Another is the possible conformational changes in prion proteins, implicated in "mad cow disease," which may be related to similar transitions in a variety of globular and fibrous proteins.

  18. Construction of a Chassis for a Tripartite Protein-Based Molecular Motor.

    PubMed

    Small, Lara S R; Bruning, Marc; Thomson, Andrew R; Boyle, Aimee L; Davies, Roberta B; Curmi, Paul M G; Forde, Nancy R; Linke, Heiner; Woolfson, Derek N; Bromley, Elizabeth H C

    2017-03-14

    Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultracentrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.

  19. Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein.

    PubMed

    Bada, M; Walther, D; Arcangioli, B; Doniach, S; Delarue, M

    2000-07-14

    Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.

  20. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    SciTech Connect

    Kutty, R. Krishnan . E-mail: kuttyk@nei.nih.gov; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-07-14

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P{sup 27}KKRKAP{sup 276}) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

  1. The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

    PubMed Central

    Durst, Kristie L.; Lutterbach, Bart; Kummalue, Tanawan; Friedman, Alan D.; Hiebert, Scott W.

    2003-01-01

    Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor. PMID:12509458

  2. Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

    SciTech Connect

    Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing; Qiu, Bingsheng; Rao, Zihe; Gao, George F.; Tien, Po

    2005-09-01

    Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion.

  3. Comparison of helix interactions in membrane and soluble alpha-bundle proteins.

    PubMed Central

    Eilers, Markus; Patel, Ashish B; Liu, Wei; Smith, Steven O

    2002-01-01

    Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with

  4. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  5. Origins of Structural Flexibility in Protein-Based Supramolecular Polymers Revealed by DEER Spectroscopy

    PubMed Central

    2015-01-01

    Modular assembly of bio-inspired supramolecular polymers is a powerful technique to develop new soft nanomaterials, and protein folding is a versatile basis for preparing such materials. Previous work demonstrated a significant difference in the physical properties of closely related supramolecular polymers composed of building blocks in which identical coiled-coil-forming peptides are cross-linked by one of two subtly different organic linkers (one flexible and the other rigid). Herein, we investigate the molecular basis for this observation by isolating a single subunit of the supramolecular polymer chain and probing its structure and conformational flexibility by double electron–electron resonance (DEER) spectroscopy. Experimental spin–spin distance distributions for two different labeling sites coupled with molecular dynamics simulations provide insights into how the linker structure impacts chain dynamics in the coiled-coil supramolecular polymer. PMID:25060334

  6. Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment

    PubMed Central

    1995-01-01

    The nuclear domain (ND)10 also described as POD or Kr bodies is involved in the development of acute promyelocytic leukemia and virus- host interactions. Immunofluorescence analysis using a variety of human autoimmune sera and monoclonal antibodies showed a typical dot like nuclear staining for ND10, suggesting that this structure consists of several proteins. Two of the ND10 proteins, Sp100 and PML are genetically characterized and show homology with several transcription factors. Here we describe NDP52, an additional novel protein of the ND10. We raised a new mAb C8A2, that specifically recognizes NDP52. Immunofluorescence analysis using this mAb showed a typical nuclear dot staining as it was described for ND10. Isolation and sequencing of the corresponding cDNA revealed that NDP52 has a predicted molecular mass of 52 kD. The deduced amino acid sequence exhibits an extended central coiled coil domain containing a leucine zipper motif. The COOH terminus of NDP52 shows homology with LIM domains, that have recently been described to mediate protein interactions, which let NDP52 appear as a suitable candidate for mediating interactions between ND10 proteins. In vivo, NDP52 is transcribed in all human tissues analyzed. Furthermore, we show that NDP52 colocalizes with the ND10 protein PML and can be redistributed upon viral infection and interferon treatment. These data suggest that ND10 proteins play an important role in the viral life cycle. PMID:7540613

  7. Orthoreovirus outer-fiber proteins are substrates for SUMO-conjugating enzyme Ubc9

    PubMed Central

    Yu, Fei; Wang, Hao; Wang, Longlong; Lu, Liqun

    2016-01-01

    Reoviruses are potential anticancer agents due to their ability to induce cell death in tumor cells. Grass carp reovirus (GCRV) is one of the best characterized models on reovirus pathogenesis in vitro. However, there is little known about how SUMOylation affects reovirus pathogenesis. The SUMO conjugating enzyme 9 (Ubc9) determines the targets of SUMOylation. Here, the protein interactions between reovirus outer fiber proteins, specifically GCRV-104 VP55, and Ubc9 were probed using a yeast two-hybrid system. The N-terminal coiled-coil domain of VP55, containing a single lysine residue, was responsible for the interaction between VP55 and Ubc9 in yeast. In solid phase binding assays, a single amino acid mutation (K87R) prevented Ubc9 from binding to VP55. Overexpression of Ubc9 enhanced GCRV-104 infection efficiency, and knockdown of Ubc9 in CIK cells inhibited viral replication, which suggested that Ubc9 was a proviral factor. Furthermore, Ubc9 was shown to bind outer fiber proteins from type II GCRV, avian reovirus and mammalian reovirus in yeast. To our knowledge, this is the first study to show that Ubc9 binds to reovirus outer-fiber proteins and likely contributes to efficient orthoreovirus replication. These results suggest that SUMOylation modifications could be targeted to improve the therapeutic efficacy of oncolytic reovirus. PMID:27806335

  8. Cell death induction by the acute promyelocytic leukemia-specific PML/RARα fusion protein

    PubMed Central

    Ferrucci, Pier Francesco; Grignani, Francesco; Pearson, Mark; Fagioli, Marta; Nicoletti, Ildo; Pelicci, Pier Giuseppe

    1997-01-01

    PML/RARα is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARα in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARα has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARα in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARα) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARα DNA binding region. Analysis of the nuclear localization of the same PML/RARα deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARα zinc fingers, is required for the proper nuclear localization of PML/RARα. We propose that the matrix-associated microspeckles are the active sites of PML/RARα and that targeting of RARα sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation. PMID:9380732

  9. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  10. Identification of a novel centrosomal protein Crp{sup F46} involved in cell cycle progression and mitosis

    SciTech Connect

    Wei Yi; Shen Enzhi; Zhao Na; Liu Qian; Fan Jinling; Marc, Jan; Wang Yongchao; Sun Le; Liang Qianjin

    2008-05-01

    A novel centrosome-related protein Crp{sup F46} was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp{sup F46} has an apparent molecular mass of {approx} 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp{sup F46} monoclonal antibody revealed that Crp{sup F46} localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp{sup F46} fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp{sup F46} knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp{sup F46} is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism.

  11. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  12. High resolution characterization of myosin IIC protein tailpiece and its effect on filament assembly.

    PubMed

    Rosenberg, Masha M; Ronen, Daniel; Lahav, Noa; Nazirov, Elvira; Ravid, Shoshana; Friedler, Assaf

    2013-04-05

    The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr(1957) or Thr(1960) inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.

  13. Obtaining structural information of small proteins using solid-state nanopores and high-bandwidth measurements

    NASA Astrophysics Data System (ADS)

    Niedzwiecki, David; Lanci, Christopher; Saven, Jeffery; Drndic, Marija

    2015-03-01

    The use of biological nanopores sensors to characterize proteins has proved a fruitful field of study. Solid-state nanopores hold several advantages over their biological counterparts, including the ability to tune pore diameter and their robustness to external conditions. Despite these advantages, the use of solid-state nanopores for protein analysis has proved difficult due to rapid translocation times of proteins and poor signal-to-noise of small peptides. Recently, improvements in high-bandwidth acquisition and in signal-to-noise have made the study of small peptides using solid-state nanopores feasible. Here we report on the detection and characterization of peptides as small as 33 amino-acids in length using sub-10 nm thin silicon nitride nanopores, giving high signal levels, combined with high-bandwidth electronics. In addition we show differentiation between monomers and dimer forms of the GCN-4 p1 leucine zipper, a coil-coil structure, and compare this with the unstructured 33-mer. The differentiation between these two forms demonstrates the possibility of extracting useful structural information from short peptide structures using modern solid-state nanopore systems.

  14. Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila

    PubMed Central

    Zhao, Hongyu; Wei, Hui; Liu, Xi; Yao, Zhenyu; Xu, Manyu; Wei, Daixu; Wang, Jiawei; Wang, Xinquan; Chen, Guo-Qiang

    2016-01-01

    Phasins or PhaPs are a group of amphiphilic proteins that are found attached to the surface of microbial polyhydroxyalkanoate (PHA) granules. They have both structural and regulatory functions and can affect intracellular PHA accumulation and mediate protein folding. The molecular basis for the diverse functions of the PhaPs has not been fully understood due to the lack of the structural knowledge. Here we report the structural and biochemical studies of the PhaP cloned from Aeromonas hydrophila (PhaPAh), which is utilized in protein and tissue engineering. The crystal structure of PhaPAh was revealed to be a tetramer with 8 α-helices adopting a coiled-coil structure. Each monomer has a hydrophobic and a hydrophilic surface, rendering the surfactant properties of the PhaPAh monomer. Based on the crystal structure, we predicted three key amino acid residues and obtained mutants with enhanced stability and improved emulsification properties. The first PhaP crystal structure, as reported in this study, is an important step towards a mechanistic understanding of how PHA is formed in vivo and why PhaP has such unique surfactant properties. At the same time, it will facilitate the study of other PhaP members that may have significant biotechnological potential as bio-surfactants and amphipathic coatings. PMID:28009010

  15. Structures and Polymorphic Interactions of Two Heptad-Repeat Regions of the SARS Virus S2 Protein

    SciTech Connect

    Deng,Y.; Liu, J.; Zheng, Q.; Yong, W.; Lu, M.

    2006-01-01

    Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct a-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.

  16. Alimentary proteins, amino acids and cholesterolemia.

    PubMed

    Blachier, François; Lancha, Antonio H; Boutry, Claire; Tomé, Daniel

    2010-01-01

    Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

  17. Electrocatalysis in proteins, nucleic acids and carbohydrates.

    PubMed

    Paleček, Emil; Bartošík, Martin; Ostatná, Veronika; Trefulka, Mojmír

    2012-02-01

    The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

  18. Crystal structure of a plectonemic RNA supercoil

    SciTech Connect

    Stagno, Jason R.; Ma, Buyong; Li, Jess; Altieri, Amanda S.; Byrd, R. Andrew; Ji, Xinhua

    2012-12-14

    Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. Here we report the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of an RNA-binding protein, two interlocking RNA coiled coils of double-stranded RNA, a 'coil of coiled coils', form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. This study provides structural insight into higher order packaging mechanisms of nucleic acids.

  19. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region.

  20. Conformations of amino acids in proteins.

    PubMed

    Hovmöller, Sven; Zhou, Tuping; Ohlson, Tomas

    2002-05-01

    The main-chain conformations of 237 384 amino acids in 1042 protein subunits from the PDB were analyzed with Ramachandran plots. The populated areas of the empirical Ramachandran plot differed markedly from the classical plot in all regions. All amino acids in alpha-helices are found within a very narrow range of phi, psi angles. As many as 40% of all amino acids are found in this most populated region, covering only 2% of the Ramachandran plot. The beta-sheet region is clearly subdivided into two distinct regions. These do not arise from the parallel and antiparallel beta-strands, which have quite similar conformations. One beta region is mainly from amino acids in random coil. The third and smallest populated area of the Ramachandran plot, often denoted left-handed alpha-helix, has a different position than that originally suggested by Ramachandran. Each of the 20 amino acids has its own very characteristic Ramachandran plot. Most of the glycines have conformations that were considered to be less favoured. These results may be useful for checking secondary-structure assignments in the PDB and for predicting protein folding.

  1. Measurement of protein using bicinchoninic acid.

    PubMed

    Smith, P K; Krohn, R I; Hermanson, G T; Mallia, A K; Gartner, F H; Provenzano, M D; Fujimoto, E K; Goeke, N M; Olson, B J; Klenk, D C

    1985-10-01

    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

  2. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

    PubMed

    Calver, A R; Michalovich, D; Testa, T T; Robbins, M J; Jaillard, C; Hill, J; Szekeres, P G; Charles, K J; Jourdain, S; Holbrook, J D; Boyfield, I; Patel, N; Medhurst, A D; Pangalos, M N

    2003-02-20

    Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

  3. The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

    PubMed Central

    Koutoulis, Anthony; Pazour, Gregory J.; Wilkerson, Curtis G.; Inaba, Kazuo; Sheng, Hong; Takada, Saeko; Witman, George B.

    1997-01-01

    We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737– 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83.4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme. PMID:9166407

  4. Identification and Characterization of a Novel Secreted Immunoglobulin Binding Protein from Group A Streptococcus

    PubMed Central

    Fagan, Peter K.; Reinscheid, Dieter; Gottschalk, Birgit; Chhatwal, Gursharan S.

    2001-01-01

    Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins of Streptococcus pyogenes are the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in the vir regulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such as Listeria monocytogenes P45 autolysin, human myosin heavy chain, macrogolgin, and Schistoma mansoni paramyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease. PMID:11447160

  5. Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

    PubMed

    Callejas-Negrete, Olga A; Plamann, Michael; Schnittker, Robert; Bartnicki-García, Salomon; Roberson, Robert W; Pimienta, Genaro; Mouriño-Pérez, Rosa R

    2015-09-01

    LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the

  6. A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence.

    PubMed Central

    Talkington, D F; Crimmins, D L; Voellinger, D C; Yother, J; Briles, D E

    1991-01-01

    PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule. Images PMID:2004810

  7. Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

    The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

  8. New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

    PubMed

    Nishimura, Kohji; Ishikawa, Syouta; Matsunami, Erika; Yamauchi, Junji; Homma, Keiichi; Faulkner, Christine; Oparka, Karl; Jisaka, Mitsuo; Nagaya, Tsutomu; Yokota, Kazushige; Nakagawa, Tsuyoshi

    2015-01-01

    Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

  9. TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

    PubMed Central

    Nwagbara, Belinda U.; Faris, Anna E.; Bearce, Elizabeth A.; Erdogan, Burcu; Ebbert, Patrick T.; Evans, Matthew F.; Rutherford, Erin L.; Enzenbacher, Tiffany B.; Lowery, Laura Anne

    2014-01-01

    Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649

  10. Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein.

    PubMed

    Moktan, Hem; Guiraldelli, Michel F; Eyster, Craig A; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R Daniel; Sung, Patrick; Zhou, Donghua H; Pezza, Roberto J

    2014-05-23

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.

  11. Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1.

    PubMed Central

    Moosmann, P; Georgiev, O; Le Douarin, B; Bourquin, J P; Schaffner, W

    1996-01-01

    Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain. PMID:9016654

  12. Protein and Amino Acid Profiles of Different Whey Protein Supplements.

    PubMed

    Almeida, Cristine C; Alvares, Thiago S; Costa, Marion P; Conte-Junior, Carlos A

    2016-01-01

    Whey protein (WP) supplements have received increasing attention by consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness of these supplements. The aim of this study was to evaluate and compare the contents of total protein (TP), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), free essential amino acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while α-LA, β-LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher (p < 0.05) concentrations of TP, α-LA, β-LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference (p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the 20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, α-LA, β-LG, and free BCAA when compared to WP-BRA.

  13. Hypochlorous and peracetic acid induced oxidation of dairy proteins.

    PubMed

    Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

    2011-02-09

    Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

  14. Flexible, symmetry-directed approach to assembling protein cages

    PubMed Central

    Sciore, Aaron; Su, Min; Koldewey, Philipp; Eschweiler, Joseph D.; Diffley, Kelsey A.; Linhares, Brian M.; Ruotolo, Brandon T.; Bardwell, James C. A.; Skiniotis, Georgios; Marsh, E. Neil G.

    2016-01-01

    The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries. PMID:27432965

  15. Flexible, symmetry-directed approach to assembling protein cages.

    PubMed

    Sciore, Aaron; Su, Min; Koldewey, Philipp; Eschweiler, Joseph D; Diffley, Kelsey A; Linhares, Brian M; Ruotolo, Brandon T; Bardwell, James C A; Skiniotis, Georgios; Marsh, E Neil G

    2016-08-02

    The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

  16. Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition*

    PubMed Central

    Uchiyama, Susumu; Kawahara, Kazuki; Hosokawa, Yuki; Fukakusa, Shunsuke; Oki, Hiroya; Nakamura, Shota; Kojima, Yukiko; Noda, Masanori; Takino, Rie; Miyahara, Yuya; Maruno, Takahiro; Kobayashi, Yuji; Ohkubo, Tadayasu; Fukui, Kiichi

    2015-01-01

    Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ∼30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition. PMID:26491021

  17. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  18. Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells▿ †

    PubMed Central

    Wu, Guikai; Lin, Yi-Tzu; Wei, Randy; Chen, Yumay; Shan, Zhiyin; Lee, Wen-Hwa

    2008-01-01

    Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules. PMID:18362163

  19. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  20. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  1. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  2. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  3. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  4. The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins.

    PubMed

    Casey, Lachlan W; Lavrencic, Peter; Bentham, Adam R; Cesari, Stella; Ericsson, Daniel J; Croll, Tristan; Turk, Dušan; Anderson, Peter A; Mark, Alan E; Dodds, Peter N; Mobli, Mehdi; Kobe, Bostjan; Williams, Simon J

    2016-10-17

    Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

  5. Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone

    PubMed Central

    Doucet, Christine M.; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

    2015-01-01

    Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests

  6. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  7. Amino acid sequences of alpha-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment.

    PubMed Central

    Gough, K H; Inglis, A S; Crewther, W G

    1978-01-01

    The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool. Images Fig. 1. PMID:697725

  8. A novel family of plant nuclear envelope-associated proteins.

    PubMed

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  9. The coarse-grained OPEP force field for non-amyloid and amyloid proteins.

    PubMed

    Chebaro, Yassmine; Pasquali, Samuela; Derreumaux, Philippe

    2012-08-02

    Coarse-grained protein models with various levels of granularity and degrees of freedom offer the possibility to explore many phenomena including folding, assembly, and recognition in terms of dynamics and thermodynamics that are inaccessible to all-atom representations in explicit aqueous solution. Here, we present a refined version of the coarse-grained optimized potential for efficient protein structure prediction (OPEP) based on a six-bead representation. The OPEP version 4.0 parameter set, which uses a new analytical formulation for the nonbonded interactions and adds specific side-chain-side-chain interactions for α-helix, is subjected to three tests. First, we show that molecular dynamics simulations at 300 K preserve the experimental rigid conformations of 17 proteins with 37-152 amino acids within a root-mean-square deviation (RMSD) of 3.1 Å after 30 ns. Extending the simulation time to 100 ns for five proteins does not change the RMSDs. Second, replica exchange molecular dynamics (REMD) simulations recover the NMR structures of three prototypical β-hairpin and α-helix peptides and the NMR three-stranded β-sheet topology of a 37-residue WW domain, starting from randomly chosen states. Third, REMD simulations on the ccβ peptide show a temperature transition from a three-stranded coiled coil to amyloid-like aggregates consistent with experiments, while simulations on low molecular weight aggregates of the prion protein helix 1 do not. Overall, these studies indicate the effectiveness of our OPEP4 coarse-grained model for protein folding and aggregation, and report two future directions for improvement.

  10. Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A.

    PubMed Central

    Sirakova, T; Kolattukudy, P E; Murwin, D; Billy, J; Leake, E; Lim, D; DeMaria, T; Bakaletz, L

    1994-01-01

    Nontypeable Haemophilus influenzae is a primary pathogen in both acute otitis media (OM) and chronic OM, yet the pathogenesis of this disease is not fully understood. Although fimbriae have been observed on all clinical OM isolates examined to date, their role in pathogenesis remains unclear. Therefore, the gene which codes for the fimbrial subunit protein (fimbrin) in nontypeable H. influenzae 1128 was isolated, cloned, and sequenced. The nucleotide sequence of the fimbrin gene was found to contain an open reading frame of 1,077 bp which would encode a mature fimbrin protein consisting of 338 amino acid with a calculated molecular mass of 36.4 kDa. The translated amino acid sequence was found to be homologous with various OmpA proteins of other gram-negative bacteria, and algorithmic analysis predicted that this protein is organized as a coiled coil. To directly test whether fimbriae are involved in pathogenesis, the fimbrin gene was disrupted, and the biological consequences of disruption were absence of both expression of the fimbrial appendage and the specific immunogold labeling thereof with antisera directed against isolated fimbrial protein, reduced adherence to human oropharyngeal cells in vitro, augmented clearance from the tympanum post-transbullar inoculation, and significantly reduced induction of OM post-intranasal inoculation in a chinchilla model compared with the fimbriated parent strain. We additionally find that either passive immunization or active immunization against isolated fimbrial protein confers partial protection against transbullar challenge. A Western blot (immunoblot) indicated a degree of serological relatedness among fimbrin proteins of 15 nontypeable and type b isolates. These data suggest that fimbrin could be useful as a component of a vaccine to protect against OM. Images PMID:7909539

  11. Multisite clickable modification of proteins using lipoic acid ligase.

    PubMed

    Plaks, Joseph G; Falatach, Rebecca; Kastantin, Mark; Berberich, Jason A; Kaar, Joel L

    2015-06-17

    Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

  12. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    PubMed

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  13. Protein and sulfur amino acid requirements of broiler breeder hens.

    PubMed

    Harms, R H; Wilson, H R

    1980-02-01

    Two experiments were conducted with Cobb color-sexed broiler breeder hens to determine their protein and sulfur amino acid requirement. A daily intake between 400 and 478 mg of methionine and between 722 and 839 mg of total sulfur amino acids was necessary for maximum egg production, the latter in a diet of 13.07% protein. Slightly lower levels supported maximum body weights. Hens laying at the highest rate consumed 23.4 g of protein per day.

  14. Los Alamos sequence analysis package for nucleic acids and proteins.

    PubMed Central

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences. PMID:6174934

  15. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  16. Kainic acid inhibits protein amino acid incorporation in select rat brain regions.

    PubMed

    Planas, A M; Soriano, M A; Ferrer, I; Rodríguez-Farré, E

    1994-11-21

    Regional incorporation of labelled methionine into proteins was studied with quantitative autoradiography in different regions of the rat brain 2.5 h following systemic kainic acid administration. Labelled protein concentration was found reduced to approximately 40% of control values in the pyramidal cell layer of hippocampus, piriform, entorhinal and perirhinal cortices, ventral lateral septum and mediodorsal thalamic nucleus. These regions showed increased levels of label not incorporated into proteins, indicating that free labelled methionine was available for protein synthesis. Reduction of protein amino acid incorporation in those brain regions selectively affected by kainic acid may be involved in subsequent tissue damage.

  17. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  18. KINETICS OF AMINO ACID INCORPORATION INTO SERUM PROTEINS

    PubMed Central

    Green, H.; Anker, H. S.

    1955-01-01

    1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q10 was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q10 of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues. PMID:13221773

  19. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  20. Amino acid composition of proteins reduces deleterious impact of mutations

    PubMed Central

    Hormoz, Sahand

    2013-01-01

    The evolutionary origin of amino acid occurrence frequencies in proteins (composition) is not yet fully understood. We suggest that protein composition works alongside the genetic code to minimize impact of mutations on protein structure. First, we propose a novel method for estimating thermodynamic stability of proteins whose sequence is constrained to a fixed composition. Second, we quantify the average deleterious impact of substituting one amino acid with another. Natural proteome compositions are special in at least two ways: 1) Natural compositions do not generate more stable proteins than the average random composition, however, they result in proteins that are less susceptible to damage from mutations. 2) Natural proteome compositions that result in more stable proteins (i.e. those of thermophiles) are also tuned to have a higher tolerance for mutations. This is consistent with the observation that environmental factors selecting for more stable proteins also enhance the deleterious impact of mutations. PMID:24108121

  1. The protein digestibility-corrected amino acid score.

    PubMed

    Schaafsma, G

    2000-07-01

    The protein digestibility-corrected amino acid score (PDCAAS) has been adopted by FAO/WHO as the preferred method for the measurement of the protein value in human nutrition. The method is based on comparison of the concentration of the first limiting essential amino acid in the test protein with the concentration of that amino acid in a reference (scoring) pattern. This scoring pattern is derived from the essential amino acid requirements of the preschool-age child. The chemical score obtained in this way is corrected for true fecal digestibility of the test protein. PDCAAS values higher than 100% are not accepted as such but are truncated to 100%. Although the principle of the PDCAAS method has been widely accepted, critical questions have been raised in the scientific community about a number of issues. These questions relate to 1) the validity of the preschool-age child amino acid requirement values, 2) the validity of correction for fecal instead of ileal digestibility and 3) the truncation of PDCAAS values to 100%. At the time of the adoption of the PDCAAS method, only a few studies had been performed on the amino acid requirements of the preschool-age child, and there is still a need for validation of the scoring pattern. Also, the scoring pattern does not include conditionally indispensable amino acids. These amino acids also contribute to the nutrition value of a protein. There is strong evidence that ileal, and not fecal, digestibility is the right parameter for correction of the amino acid score. The use of fecal digestibility overestimates the nutritional value of a protein, because amino acid nitrogen entering the colon is lost for protein synthesis in the body and is, at least in part, excreted in urine as ammonia. The truncation of PDCAAS values to 100% can be defended only for the limited number of situations in which the protein is to be used as the sole source of protein in the diet. For evaluation of the nutritional significance of proteins as

  2. Protein packing: dependence on protein size, secondary structure and amino acid composition.

    PubMed

    Fleming, P J; Richards, F M

    2000-06-02

    We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.

  3. Re-evaluation of turbidimetry of proteins by use of aromatic sulfonic acids and chloroacetic acids.

    PubMed

    Ebina, S; Nagai, Y

    1979-02-01

    From studies on 11 different proteins (including native albumin and albumin with reduced disulfide-bridges) treated with sulfosalicylic, 2-naphthalenesulfonic, toluenesulfonic, dichloroacetic, or trichloroacetic acids, we elucidate the interactions determining the resulting turbidities and other factors affecting turbidities, and we discuss the clinical utility of such turbidimetry. At least three interactions are important in determining turbidity: reduction of positive charges on the protein, hydrogen bonding of the non-ionized chloroacetic acids with the protein, and hydrophobic interaction of the aromatic sulfonic acids with albumin. Turbidity varies appreciably with the species of acid and protein, concentrations of acid, temperature, and standing time after acid is added. We conclude that this technique should be restricted to confirming proteinuria.

  4. Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement.

    PubMed

    Oikawa, Kazusato; Yamasato, Akihiro; Kong, Sam-Geun; Kasahara, Masahiro; Nakai, Masato; Takahashi, Fumio; Ogura, Yasunobu; Kagawa, Takatoshi; Wada, Masamitsu

    2008-10-01

    Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

  5. BIOACTIVE PROTEINS, PEPTIDES, AND AMINO ACIDS FROM MACROALGAE(1).

    PubMed

    Harnedy, Pádraigín A; FitzGerald, Richard J

    2011-04-01

    Macroalgae are a diverse group of marine organisms that have developed complex and unique metabolic pathways to ensure survival in highly competitive marine environments. As a result, these organisms have been targeted for mining of natural biologically active components. The exploration of marine organisms has revealed numerous bioactive compounds that are proteinaceous in nature. These include proteins, linear peptides, cyclic peptides and depsipeptides, peptide derivatives, amino acids, and amino acid-like components. Furthermore, some species of macroalgae have been shown to contain significant levels of protein. While some protein-derived bioactive peptides have been characterized from macroalgae, macroalgal proteins currently still represent good candidate raw materials for biofunctional peptide mining. This review will provide an overview of the important bioactive amino-acid-containing compounds that have been identified in macroalgae. Moreover, the potential of macroalgal proteins as substrates for the generation of biofunctional peptides for utilization as functional foods to provide specific health benefits will be discussed.

  6. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  7. Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

    PubMed Central

    Linstedt, A D; Mehta, A; Suhan, J; Reggio, H; Hauri, H P

    1997-01-01

    It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting. Images PMID:9201717

  8. Protein turnover, amino acid profile and amino acid flux in juvenile shrimp Litopenaeus vannamei: effects of dietary protein source.

    PubMed

    Mente, Eleni; Coutteau, Peter; Houlihan, Dominic; Davidson, Ian; Sorgeloos, Patrick

    2002-10-01

    The effect of dietary protein on protein synthesis and growth of juvenile shrimps Litopenaeus vannamei was investigated using three different diets with equivalent protein content. Protein synthesis was investigated by a flooding dose of tritiated phenylalanine. Survival, specific growth and protein synthesis rates were higher, and protein degradation was lower, in shrimps fed a fish/squid/shrimp meal diet, or a 50% laboratory diet/50% soybean meal variant diet, than in those fed a casein-based diet. The efficiency of retention of synthesized protein as growth was 94% for shrimps fed the fish meal diet, suggesting a very low protein turnover rate; by contrast, the retention of synthesized protein was only 80% for shrimps fed the casein diet. The amino acid profile of the casein diet was poorly correlated with that of the shrimps. 4 h after a single meal the protein synthesis rates increased following an increase in RNA activity. A model was developed for amino acid flux, suggesting that high growth rates involve a reduction in the turnover of proteins, while amino acid loss appears to be high.

  9. Design and construction of self-assembling supramolecular protein complexes using artificial and fusion proteins as nanoscale building blocks.

    PubMed

    Kobayashi, Naoya; Arai, Ryoichi

    2017-02-01

    The central goal of nanobiotechnology is to design and construct novel biomaterials of nanometer sizes. In this short review, we describe recent progress of several approaches for designing and creating artificial self-assembling protein complexes and primarily focus on the following biotechnological strategies for using artificial and fusion proteins as nanoscale building blocks: fusion proteins designed for symmetrical self-assembly; three-dimensional domain-swapped oligomers; self-assembling designed coiled-coil peptide modules; metal-directed self-assembling engineered proteins; computationally designed self-assembling de novo proteins; and self-assembling protein nanobuilding blocks (PN-Blocks) using an intermolecularly folded dimeric de novo protein. These state-of-the-art nanobiotechnologies for designing supramolecular protein complexes will facilitate the development of novel functional nanobiomaterials.

  10. Mitotic apparatus: the selective extraction of protein with mild acid.

    PubMed

    Bibring, T; Baxandall, J

    1968-07-26

    The treatment of isolated mitotic apparatus with mild (pH 3) hydrochloric acid results in the extraction of less than 10 percent of its protein, accompanied by the selective morphological disappearance of the microtubules. The same extraction can be shown to dissolve outer doublet microtubules from sperm flagella. A protein with points of similarity to the flagellar microtubule protein is the major component of the extract from mitotic apparatus.

  11. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  12. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    PubMed

    Blatti, Jillian L; Beld, Joris; Behnke, Craig A; Mendez, Michael; Mayfield, Stephen P; Burkart, Michael D

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  13. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  14. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  15. Evolution of hard proteins in the sauropsid integument in relation to the cornification of skin derivatives in amniotes

    PubMed Central

    Alibardi, Lorenzo; Valle, Luisa Dalla; Nardi, Alessia; Toni, Mattia

    2009-01-01

    Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages probably derived from the localization of specialized areas of dermal–epidermal interaction in the integument. The horny scales and the other derivatives were formed from large areas of dermal–epidermal interaction. The evolution of these skin appendages was characterized by the production of specific coiled-coil keratins and associated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsids contain a double beta-folded sequence of about 20 amino acids, known as the core-box. The core-box shows 60%–95% sequence identity with known reptilian and avian proteins. The core-box determines the polymerization of these proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequences for these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolution in reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestral glycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrix proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral protein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently in the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high glycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, which evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In therapsids-mammals, mutations of the ancestral proteins

  16. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  17. Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.

    PubMed

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-08-08

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M(-1) s(-1)) were significantly higher than that measured for H2O2 (1.5 M(-1) s(-1)). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5 × 10(3) M(-1) s(-1). Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.

  18. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  19. Plasma amino acid response to graded levels of escape protein.

    PubMed

    Gibb, D J; Klopfenstein, T J; Britton, R A; Lewis, A J

    1992-09-01

    A trial was conducted to examine the potential of using plasma amino acid responses to graded levels of escape protein to determine limiting amino acids in cattle. Growing calves (n = 120; mean BW = 220 +/- 21 kg) were fed a basal diet of corncob:sorghum silage (61:39) and were individually supplemented with distillers' dried grains (DDG), heat-damaged DDG (H-DDG), feather meal (FTH), or urea. The urea supplement was mixed with DDG and H-DDG to allow 0, 20, 35, 50, 65, or 80% of the supplemental CP to come from distillers' protein and maintain an 11.5% CP diet. Urea supplement was mixed with FTH to allow 0, 22, 39, 56, 73, or 90% of the supplemental CP to come from FTH. Dietary CP ranged from 11.5% at the 0% level to 17.3% at the 90% level. Plasma concentration of most essential plasma amino acids responded (P less than .10) linearly and(or) quadratically to increased escape protein. The broken-line response of plasma methionine at low DDG intake suggested that methionine was limiting at low levels of escape protein. An initial decrease followed by a plateau fit by a broken line indicated that histidine became limiting in FTH diets, and lysine eventually became limiting for DDG, H-DDG, and FTH diets before maximum BW gain was reached. Results indicate that plasma amino acid responses may identify amino acids that become limiting with increasing escape protein.

  20. A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components.

    PubMed

    de Leon, Jessica Cruz; Scheumann, Nicole; Beatty, Wandy; Beck, Josh R; Tran, Johnson Q; Yau, Candace; Bradley, Peter J; Gull, Keith; Wickstead, Bill; Morrissette, Naomi S

    2013-07-01

    SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

  1. Hearing in Drosophila Requires TilB, a Conserved Protein Associated With Ciliary Motility

    PubMed Central

    Kavlie, Ryan G.; Kernan, Maurice J.; Eberl, Daniel F.

    2010-01-01

    Cilia were present in the earliest eukaryotic ancestor and underlie many biological processes ranging from cell motility and propulsion of extracellular fluids to sensory physiology. We investigated the contribution of the touch insensitive larva B (tilB) gene to cilia function in Drosophila melanogaster. Mutants of tilB exhibit dysfunction in sperm flagella and ciliated dendrites of chordotonal organs that mediate hearing and larval touch sensitivity. Mutant sperm axonemes as well as sensory neuron dendrites of Johnston's organ, the fly's auditory organ, lack dynein arms. Through deficiency mapping and sequencing candidate genes, we identified tilB mutations in the annotated gene CG14620. A genomic CG14620 transgene rescued deafness and male sterility of tilB mutants. TilB is a 395-amino-acid protein with a conserved N-terminal leucine-rich repeat region at residues 16–164 and a coiled-coil domain at residues 171–191. A tilB-Gal4 transgene driving fluorescently tagged TilB proteins elicits cytoplasmic expression in embryonic chordotonal organs, in Johnston's organ, and in sperm flagella. TilB does not appear to affect tubulin polyglutamylation or polyglycylation. The phenotypes and expression of tilB indicate function in cilia construction or maintenance, but not in intraflagellar transport. This is also consistent with phylogenetic association of tilB homologs with presence of genes encoding axonemal dynein arm components. Further elucidation of tilB functional mechanisms will provide greater understanding of cilia function and will facilitate understanding ciliary diseases. PMID:20215474

  2. HOMER2, a Stereociliary Scaffolding Protein, Is Essential for Normal Hearing in Humans and Mice

    PubMed Central

    Azaiez, Hela; Shearer, A. Eliot; Huygen, Patrick L. M.; Bu, Fengxiao; Hildebrand, Michael S.; Ranum, Paul T.; Shibata, Seiji B.; Turner, Ann; Zhang, Yuzhou; Kimberling, William J.; Cornell, Robert A.; Smith, Richard J. H.

    2015-01-01

    Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS) the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL). After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action. PMID:25816005

  3. Stimulation of nonselective amino acid export by glutamine dumper proteins.

    PubMed

    Pratelli, Réjane; Voll, Lars M; Horst, Robin J; Frommer, Wolf B; Pilot, Guillaume

    2010-02-01

    Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

  4. Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

    SciTech Connect

    Martin-Garcia, Fernando; Mendieta-Moreno, Jesus Ignacio; Mendieta, Jesus

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. Black-Right-Pointing-Pointer HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. Black-Right-Pointing-Pointer HRS domains of F protein form three single {alpha}-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from {beta}-sheet conformation to an elongated coil and then spontaneously to an {alpha}-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.

  5. The multiple roles of fatty acid handling proteins in brain

    PubMed Central

    Moullé, Valentine S. F.; Cansell, Céline; Luquet, Serge; Cruciani-Guglielmacci, Céline

    2012-01-01

    Lipids are essential components of a living organism as energy source but also as constituent of the membrane lipid bilayer. In addition fatty acid (FA) derivatives interact with many signaling pathways. FAs have amphipathic properties and therefore require being associated to protein for both transport and intracellular trafficking. Here we will focus on several FA handling proteins, among which the fatty acid translocase/CD36 (FAT/CD36), members of fatty acid transport proteins (FATPs), and lipid chaperones fatty acid-binding proteins (FABPs). A decade of extensive studies has helped decipher the mechanism of action of these proteins in peripheral tissue with high lipid metabolism. However, considerably less information is available regarding their role in the brain, despite the high lipid content of this tissue. This review will primarily focus on the recent studies that have highlighted the crucial role of lipid handling proteins in brain FA transport, neuronal differentiation and development, cognitive processes and brain diseases. Finally a special focus will be made on the recent studies that have revealed the role of FAT/CD36 in brain lipid sensing and nervous control of energy balance. PMID:23060810

  6. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  7. Tuning assembly size in Peptide-based supramolecular polymers by modulation of subunit association affinity.

    PubMed

    Oshaben, Kaylyn M; Horne, W Seth

    2014-04-14

    Nature uses proteins and nucleic acids to form a wide array of functional architectures, and scientists have found inspiration from these structures in the rational design of synthetic biomaterials. We have recently shown that a modular subunit consisting of two α-helical coiled coil peptides attached at their midpoints by an organic linking group can spontaneously self-assemble in aqueous solution to form a soluble supramolecular polymer. Here we explore the use of coiled-coil association affinity, readily tuned by amino acid sequence, as a means to predictably alter properties of these supramolecular assemblies. A series of dimeric coiled-coil peptide sequences with identical quaternary folded structures but systematically altered folded stability were designed and biophysically characterized. The sequences were cross-linked to generate a series of branched, self-assembling biomacromolecular subunits. A clear relationship is observed between coiled-coil association affinity and apparent hydrodynamic diameter of the supramolecular polymers formed by these subunits. Our results provide a family of soluble supramolecular polymers of tunable size and well-characterized coiled-coil sequences that add to the library of building blocks available for use in the rational design of protein-based supramolecular biomaterials.

  8. TRIM proteins as RING finger E3 ubiquitin ligases.

    PubMed

    Ikeda, Kazuhiro; Inoue, Satoshi

    2012-01-01

    The tripartite motif(TRIM) proteins harboring the RING finger, B-box and coiled-coil (RBCC) domain motifs form a large protein family. The members of this family are involved in various biological processes, including growth, differentiation, apoptosis and transcription and also in diseases and oncogenesis. Recent studies have revealed that TRIM proteins play key roles in innate antiviral immunity. An accumulating body of evidence has demonstrated that some TRIM proteins function as E3 ubiquitin ligases in specific ubiquitin-mediated protein degradation pathways; however, the precise mechanisms underlying this function have not been fully elucidated. In this chapter, we focus on the TRIM family of proteins specially with regard to E3 ligase.

  9. Roles of intrinsic disorder in protein-nucleic acid interactions.

    PubMed

    Dyson, H Jane

    2012-01-01

    Interactions between proteins and nucleic acids typify the role of disordered segments, linkers, tails and other entities in the function of complexes that must form with high affinity and specificity but which must be capable of dissociating when no longer needed. While much of the emphasis in the literature has been on the interactions of disordered proteins with other proteins, disorder is also frequently observed in nucleic acids (particularly RNA) and in the proteins that interact with them. The interactions of disordered proteins with DNA most often manifest as molding of the protein onto the B-form DNA structure, although some well-known instances involve remodeling of the DNA structure that seems to require that the interacting proteins be disordered to various extents in the free state. By contrast, induced fit in RNA-protein interactions has been recognized for many years-the existence and prevalence of this phenomenon provides the clearest possible evidence that RNA and its interactions with proteins must be considered as highly dynamic, and the dynamic nature of RNA and its multiplicity of folded and unfolded states is an integral part of its nature and function.

  10. The C-Terminal Portion of the Hrs Protein Interacts with Tsg101 and Interferes with Human Immunodeficiency Virus Type 1 Gag Particle Production▿

    PubMed Central

    Bouamr, Fadila; Houck-Loomis, Brian R.; De Los Santos, Martha; Casaday, Rebecca J.; Johnson, Marc C.; Goff, Stephen P.

    2007-01-01

    The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in “lump-like” structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein. PMID:17182674

  11. A general method of protein purification for recombinant unstructured non-acidic proteins.

    PubMed

    Campos, Francisco; Guillén, Gabriel; Reyes, José L; Covarrubias, Alejandra A

    2011-11-01

    Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

  12. Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein.

    PubMed

    Lee, Duck Yeon; Kim, Kyeong-Ae; Yu, Yeon Gyu; Kim, Key-Sun

    2004-07-30

    Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.

  13. Comparative proteomic analysis of differentially expressed proteins in β-aminobutyric acid enhanced Arabidopsis thaliana tolerance to simulated acid rain.

    PubMed

    Liu, Tingwu; Jiang, Xinwu; Shi, Wuliang; Chen, Juan; Pei, Zhenming; Zheng, Hailei

    2011-05-01

    Acid rain is a worldwide environmental issue that has seriously destroyed forest ecosystems. As a highly effective and broad-spectrum plant resistance-inducing agent, β-aminobutyric acid could elevate the tolerance of Arabidopsis when subjected to simulated acid rain. Using comparative proteomic strategies, we analyzed 203 significantly varied proteins of which 175 proteins were identified responding to β-aminobutyric acid in the absence and presence of simulated acid rain. They could be divided into ten groups according to their biological functions. Among them, the majority was cell rescue, development and defense-related proteins, followed by transcription, protein synthesis, folding, modification and destination-associated proteins. Our conclusion is β-aminobutyric acid can lead to a large-scale primary metabolism change and simultaneously activate antioxidant system and salicylic acid, jasmonic acid, abscisic acid signaling pathways. In addition, β-aminobutyric acid can reinforce physical barriers to defend simulated acid rain stress.

  14. Peptide–oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    PubMed Central

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte S.; Thomsen, Rasmus P.; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W.; Wengel, Jesper; Jensen, Knud J.

    2016-01-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design. PMID:27464951

  15. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    NASA Astrophysics Data System (ADS)

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte S.; Thomsen, Rasmus P.; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W.; Wengel, Jesper; Jensen, Knud J.

    2016-07-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

  16. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

    PubMed

    Lou, Chenguang; Martos-Maldonado, Manuel C; Madsen, Charlotte S; Thomsen, Rasmus P; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W; Wengel, Jesper; Jensen, Knud J

    2016-07-28

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

  17. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  18. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  19. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  20. Streptococcal M protein: molecular design and biological behavior.

    PubMed Central

    Fischetti, V A

    1989-01-01

    M protein is a major virulence determinant for the group A streptococcus by virtue of its ability to allow the organism to resist phagocytosis. Common in eucaryotes, the fibrillar coiled-coil design for the M molecule may prove to be a common motif for surface proteins in gram-positive organisms. This type of structure offers the organism several distinct advantages, ranging from antigenic variation to multiple functional domains. The close resemblance of this molecular design to that of certain mammalian proteins could help explain on a molecular level the formation of epitopes responsible for serological cross-reactions between microbial and mammalian proteins. Many of the approaches described in the elucidation of the M-protein structure may be applied for characterizing similar molecules in other microbial systems. Images PMID:2670192

  1. Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

    PubMed

    Gregory, Katherine E; Winston, Abigail B; Yamamoto, Hidemi S; Dawood, Hassan Y; Fashemi, Titilayo; Fichorova, Raina N; Van Marter, Linda J

    2014-06-01

    Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

  2. [Photochemistry and UV Spectroscopy of Proteins and Nucleic Acids].

    PubMed

    Wierzchowski, Kazimierz Lech

    2015-01-01

    The article presents a short history of David Shugar studies in the field of photochemistry and UV spectroscopy of proteins and nucleic acids, carried out since the late 1940s. to the beginning of the 1970s. of the 20th century, with some references to the state of related research in those days.

  3. Amino acid nutrition beyond methionine and lysine for milk protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  4. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

    PubMed

    Xu, Y J; Yau, L; Yu, L P; Elimban, V; Zahradka, P; Dhalla, N S

    1996-12-13

    Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

  5. Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus.

    PubMed

    Gao, Jing; Lu, Guangwen; Qi, Jianxun; Li, Yan; Wu, Ying; Deng, Yao; Geng, Heyuan; Li, Hongbin; Wang, Qihui; Xiao, Haixia; Tan, Wenjie; Yan, Jinghua; Gao, George F

    2013-12-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.

  6. CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

    PubMed Central

    Jansen, Jos C.; Cirak, Sebahattin; van Scherpenzeel, Monique; Timal, Sharita; Reunert, Janine; Rust, Stephan; Pérez, Belén; Vicogne, Dorothée; Krawitz, Peter; Wada, Yoshinao; Ashikov, Angel; Pérez-Cerdá, Celia; Medrano, Celia; Arnoldy, Andrea; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; Quelhas, Dulce; Diogo, Luisa; Rymen, Daisy; Jaeken, Jaak; Guffon, Nathalie; Cheillan, David; van den Heuvel, Lambertus P.; Maeda, Yusuke; Kaiser, Olaf; Schara, Ulrike; Gerner, Patrick; van den Boogert, Marjolein A.W.; Holleboom, Adriaan G.; Nassogne, Marie-Cécile; Sokal, Etienne; Salomon, Jody; van den Bogaart, Geert; Drenth, Joost P.H.; Huynen, Martijn A.; Veltman, Joris A.; Wevers, Ron A.; Morava, Eva; Matthijs, Gert; Foulquier, François; Marquardt, Thorsten; Lefeber, Dirk J.

    2016-01-01

    Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal

  7. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  8. Kinetic study of sulphuric acid hydrolysis of protein feathers.

    PubMed

    Ben Hamad Bouhamed, Sana; Kechaou, Nabil

    2017-02-28

    Poultry feather keratin is the most important by-product from the poultry industry due to its abundance. Different methods have been still applied to process this by-product such as enzymatic hydrolysis which is expensive and inapplicable at the industrial level. This paper presents a study of acid hydrolysis of poultry feathers using different types of acids, sulphuric acid concentration, different temperatures and solid to liquid ratio to obtain a liquid product rich in peptides. The feathers analysis revealed a crude protein content of 88.83%. A maximum peptides production of 676 mg/g was reached using sulphuric acid, 1 molar acid concentration and 50 g/l solid to liquid ratio at a temperature of 90 °C after 300 min. A reaction scheme for protein aggregation and decomposition to polypeptides and amino acids was proposed and a kinetic model for peptides production was developed. The proposed kinetic model proved to be well adapted to the experimental data with R (2) = 0.99.

  9. Protein folding trajectories can be described quantitatively by one-dimensional diffusion over measured energy landscapes

    NASA Astrophysics Data System (ADS)

    Neupane, Krishna; Manuel, Ajay P.; Woodside, Michael T.

    2016-07-01

    Protein folding features a diffusive search over a multidimensional energy landscape in conformational space for the minimum-energy structure. Experiments, however, are usually interpreted in terms of a one-dimensional (1D) projection of the full landscape onto a practical reaction coordinate. Although simulations have shown that folding kinetics can be described well by diffusion over a 1D projection, 1D approximations have not yet been fully validated experimentally. We used folding trajectories of single molecules held under tension in optical tweezers to compare the conditional probability of being on a transition path, calculated from the trajectory, with the prediction for ideal 1D diffusion over the measured 1D landscape, calculated from committor statistics. We found good agreement for the protein PrP (refs ,) and for one of the structural transitions in a leucine-zipper coiled-coil, but not for a second transition in the coiled-coil, owing to poor reaction-coordinate quality. These results show that 1D descriptions of folding can indeed be good, even for complex tertiary structures. More fundamentally, they also provide a fully experimental validation of the basic physical picture of folding as diffusion over a landscape.

  10. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.

  11. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  12. A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement

    PubMed Central

    Valuchova, Sona; Fulnecek, Jaroslav; Petrov, Alexander P.; Tripsianes, Konstantinos; Riha, Karel

    2016-01-01

    Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions. PMID:28008962

  13. Dietary protein's and dietary acid load's influence on bone health.

    PubMed

    Remer, Thomas; Krupp, Danika; Shi, Lijie

    2014-01-01

    A variety of genetic, mechano-response-related, endocrine-metabolic, and nutritional determinants impact bone health. Among the nutritional influences, protein intake and dietary acid load are two of the factors most controversially discussed. Although in the past high protein intake was often assumed to exert a primarily detrimental impact on bone mass and skeletal health, the majority of recent studies indicates the opposite and suggests a bone-anabolic influence. Studies examining the influence of alkalizing diets or alkalizing supplement provision on skeletal outcomes are less consistent, which raises doubts about the role of acid-base status in bone health. The present review critically evaluates relevant key issues such as acid-base terminology, influencing factors of intestinal calcium absorption, calcium balance, the endocrine-metabolic milieu related to metabolic acidosis, and some methodological aspects of dietary exposure and bone outcome examinations. It becomes apparent that for an adequate identification and characterization of either dietary acid load's or protein's impact on bone, the combined assessment of both nutritional influences is necessary.

  14. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  15. Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

    PubMed

    Portovedo, Mariana; Ignacio-Souza, Letícia M; Bombassaro, Bruna; Coope, Andressa; Reginato, Andressa; Razolli, Daniela S; Torsoni, Márcio A; Torsoni, Adriana S; Leal, Raquel F; Velloso, Licio A; Milanski, Marciane

    2015-01-01

    Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

  16. Electricity-free, sequential nucleic acid and protein isolation.

    PubMed

    Pawlowski, David R; Karalus, Richard J

    2012-05-15

    Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification

  17. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

    PubMed

    van Hemert, Martijn J; Lamers, Gerda E M; Klein, Dionne C G; Oosterkamp, Tjerk H; Steensma, H Yde; van Heusden, G Paul H

    2002-04-16

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

  18. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies

    PubMed Central

    van Hemert, Martijn J.; Lamers, Gerda E. M.; Klein, Dionne C. G.; Oosterkamp, Tjerk H.; Steensma, H. Yde; van Heusden, G. Paul H.

    2002-01-01

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. PMID:11929974

  19. Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Zhang, BinBin; Zheng, Dan; Al-Rubeaan, Khalid; Luong, John H T; Sheu, Fwu-Shan

    2011-10-01

    This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu(2+) in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.

  20. Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

    PubMed

    Pattison, David I; Hawkins, Clare L; Davies, Michael J

    2007-08-28

    Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

  1. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  2. Autophagy-related protein MoAtg14 is involved in differentiation, development and pathogenicity in the rice blast fungus Magnaporthe oryzae

    PubMed Central

    Liu, Xiao-Hong; Zhao, Ya-Hui; Zhu, Xue-Ming; Zeng, Xiao-Qing; Huang, Lu-Yao; Dong, Bo; Su, Zhen-Zhu; Wang, Yao; Lu, Jian-Ping; Lin, Fu-Cheng

    2017-01-01

    Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the vacuole/lysosome in eukaryotic cells. MoAtg14 in M. oryzae, a hitherto uncharacterized protein, is the highly divergent homolog of the yeast Atg14 and the mammal BARKOR. The MoATG14 deletion mutant exhibited collapse in the center of the colonies, poor conidiation and a complete loss of virulence. Significantly, the ΔMoatg14 mutant showed delayed breakdown of glycogen, less lipid bodies, reduced turgor pressure in the appressorium and impaired conidial autophagic cell death. The autophagic process was blocked in the ΔMoatg14 mutant, and the autophagic degradation of the marker protein GFP-MoAtg8 was interrupted. GFP-MoAtg14 co-localized with mCherry-MoAtg8 in the aerial hypha. In addition, a conserved coiled-coil domain was predicted in the N-terminal region of the MoAtg14 protein, a domain which could mediate the interaction between MoAtg14 and MoAtg6. The coiled-coil domain of the MoAtg14 protein is essential for its function in autophagy and pathogenicity. PMID:28067330

  3. Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression.

    PubMed

    Yang, Jun; Staples, Oliver; Thomas, Luke W; Briston, Thomas; Robson, Mathew; Poon, Evon; Simões, Maria L; El-Emir, Ethaar; Buffa, Francesca M; Ahmed, Afshan; Annear, Nicholas P; Shukla, Deepa; Pedley, Barbara R; Maxwell, Patrick H; Harris, Adrian L; Ashcroft, Margaret

    2012-02-01

    Increased expression of the regulatory subunit of HIFs (HIF-1α or HIF-2α) is associated with metabolic adaptation, angiogenesis, and tumor progression. Understanding how HIFs are regulated is of intense interest. Intriguingly, the molecular mechanisms that link mitochondrial function with the HIF-regulated response to hypoxia remain to be unraveled. Here we describe what we believe to be novel functions of the human gene CHCHD4 in this context. We found that CHCHD4 encodes 2 alternatively spliced, differentially expressed isoforms (CHCHD4.1 and CHCHD4.2). CHCHD4.1 is identical to MIA40, the homolog of yeast Mia40, a key component of the mitochondrial disulfide relay system that regulates electron transfer to cytochrome c. Further analysis revealed that CHCHD4 proteins contain an evolutionarily conserved coiled-coil-helix-coiled-coil-helix (CHCH) domain important for mitochondrial localization. Modulation of CHCHD4 protein expression in tumor cells regulated cellular oxygen consumption rate and metabolism. Targeting CHCHD4 expression blocked HIF-1α induction and function in hypoxia and resulted in inhibition of tumor growth and angiogenesis in vivo. Overexpression of CHCHD4 proteins in tumor cells enhanced HIF-1α protein stabilization in hypoxic conditions, an effect insensitive to antioxidant treatment. In human cancers, increased CHCHD4 expression was found to correlate with the hypoxia gene expression signature, increasing tumor grade, and reduced patient survival. Thus, our study identifies a mitochondrial mechanism that is critical for regulating the hypoxic response in tumors.

  4. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    PubMed

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.

  5. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  6. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  7. ARG1 (altered response to gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton

    NASA Technical Reports Server (NTRS)

    Sedbrook, J. C.; Chen, R.; Masson, P. H.

    1999-01-01

    Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenorhabditis elegans suggest that ARG1 is involved in other essential processes.

  8. Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

    PubMed Central

    Henriksen, Svend B.; Arnason, Omar; Söring, Jón; Petersen, Steffen B.

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  9. The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15.

    PubMed Central

    Sengar, A S; Wang, W; Bishay, J; Cohen, S; Egan, S E

    1999-01-01

    Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components. PMID:10064583

  10. Identification of a new protein in the centrosome-like "atractophore" of Trichomonas vaginalis.

    PubMed

    Bricheux, Geneviève; Coffe, Gérard; Brugerolle, Guy

    2007-06-01

    The human parasite Trichomonas vaginalis has specific structural bodies, atractophores, associated at one end to the kinetosomes and at the other to the spindle during division. A monoclonal antibody specific for a component of this structure was obtained. It recognizes a protein with a predicted molecular mass of 477 kDa. Sequence analysis of this protein shows that P477 belongs to the family of large coiled-coil proteins, sharing a highly versatile protein folding motif adaptable to many biological functions. P477-might act as an anchor to localize cellular activities and components to the golgi centrosomal region. It may represent a new class of structural proteins, since similar proteins were found in many protozoans.

  11. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid.

  12. Longitudinal evolution of true protein, amino acids and bioactive proteins in breast milk: a developmental perspective.

    PubMed

    Lönnerdal, Bo; Erdmann, Peter; Thakkar, Sagar K; Sauser, Julien; Destaillats, Frédéric

    2017-03-01

    The protein content of breast milk provides a foundation for estimating protein requirements of infants. Because it serves as a guideline for regulatory agencies issuing regulations for infant formula composition, it is critical that information on the protein content of breast milk is reliable. We have therefore carried out a meta-analysis of the protein and amino acid contents of breast milk and how they evolve during lactation. As several bioactive proteins are not completely digested in the infant and therefore represent "non-utilizable" protein, we evaluated the quantity, mechanism of action and digestive fate of several major breast milk proteins. A better knowledge of the development of the protein contents of breast milk and to what extent protein utilization changes with age of the infant will help improve understanding of protein needs in infancy. It is also essential when designing the composition of infant formulas, particularly when the formula uses a "staging" approach in which the composition of the formula is modified in stages to reflect changes in breast milk and changing requirements as the infant ages.

  13. Phytic acid reduction in soy protein improves zinc bioavailability

    SciTech Connect

    Zhou, J.R.; Wong, M.S.; Burns, R.A.; Erdman, J.W. Jr. Mead Johnson Research Center, Evansville, IN )

    1991-03-15

    The objective of this study was to confirm previous studies that have suggested that reduction of phytic acid in soy improved zinc bioavailability (BV). Two commercially-produced soybean isolates containing either a normal phytic acid level or a reduced level were formulated into diets so as to provide 6 or 9 ppm zinc. Control diets were egg white protein-based and contained 3, 6 or 9 ppm zinc from zinc carbonate. Weanling male rats were fed these diets for 21 days and food intake and weight gain monitored. Slope ratio analysis of total tibia zinc content compared to total zinc intake revealed that zinc BV from reduced phytic acid soy isolate-containing diets was indistinguishable from control egg white diets. In contrast, zinc BV from normal soy isolate diets was significantly reduced compared to reduced phytic acid and control diets. These results coupled with other results indicate that phytic acid is the inhibitory factor in soybean products that results in reduced zinc BV.

  14. The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis.

    PubMed

    Samejima, Kumiko; Platani, Melpomeni; Wolny, Marcin; Ogawa, Hiromi; Vargiu, Giulia; Knight, Peter J; Peckham, Michelle; Earnshaw, William C

    2015-08-28

    The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

  15. Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.

    PubMed

    Engel, Jürgen

    2004-06-01

    Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.

  16. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  17. Dynamics of palmitic acid complexed with rat intestinal fatty acid binding protein.

    PubMed

    Zhu, L; Kurian, E; Prendergast, F G; Kemple, M D

    1999-02-02

    Dynamics of palmitic acid (PA), isotopically enriched with 13C at the second, seventh, or terminal methyl position, were investigated by 13C NMR. Relaxation measurements were made on PA bound to recombinant rat intestinal fatty acid binding protein (I-FABP) at pH 5.5 and 23 degreesC, and, for comparison, on PA incorporated into 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) micelles, and dissolved in methanol. The 13C relaxation data, T1, and steady-state nuclear Overhauser effect (NOE) obtained at two different magnetic fields were interpreted using the model-free approach [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall rotational correlation time of the fatty acid.protein complex was 2.5 +/- 0.4 ns, which is substantially less than the value expected for the protein itself (>6 ns). Order parameters (S2), which are a measure of the amplitude of the internal motion of individual C-H vectors with respect to the PA molecule, while largest for C-2 and smallest for the methyl carbon, were relatively small (<0.4) in the protein complex. S2 values for given C-H vectors also were smaller for PA in the MPPC micelles and in methanol than in the protein complex. Correlation times reflective of the time scale of the internal motion of the C-H vectors were in all cases <60 ps. These results support the view that the fatty acid is not rigidly anchored within the I-FABP binding pocket, but rather has considerable freedom to move within the pocket.

  18. Minimal genome encoding proteins with constrained amino acid repertoire

    PubMed Central

    Tsoy, Olga; Yurieva, Marina; Kucharavy, Andrey; O'Reilly, Mary; Mushegian, Arcady

    2013-01-01

    Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging. PMID:23873957

  19. Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

    PubMed Central

    Dib-Hajj, F.; Khan, R.; Giedroc, D. P.

    1993-01-01

    The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer. PMID:8443601

  20. Crystallization of a paraspeckle protein PSPC1-NONO heterodimer.

    PubMed

    Passon, Daniel M; Lee, Mihwa; Fox, Archa H; Bond, Charles S

    2011-10-01

    The paraspeckle component 1 (PSPC1) and non-POU-domain-containing octamer-binding protein (NONO) heterodimer is an essential structural component of paraspeckles, ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. PSPC1 and NONO both belong to the Drosophila behaviour and human splicing (DBHS) protein family, which has been implicated in many aspects of RNA processing. A heterodimer of the core DBHS conserved region of PSPC1 and NONO comprising two tandemly arranged RNA-recognition motifs (RRMs), a NONA/paraspeckle (NOPS) domain and part of a predicted coiled-coil domain has been crystallized in space group C2, with unit-cell parameters a = 90.90, b = 67.18, c = 94.08 Å, β = 99.96°. The crystal contained one heterodimer in the asymmetric unit and diffracted to 1.9 Å resolution using synchrotron radiation.

  1. Truncated variants of hyaluronan-binding protein 1 bind hyaluronan and induce identical morphological aberrations in COS-1 cells.

    PubMed Central

    Sengupta, Aniruddha; Tyagi, Rakesh K; Datta, Kasturi

    2004-01-01

    Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between alpha-helices at its N- and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the alpha-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network. PMID:15005653

  2. An amino acid code for irregular and mixed protein packing.

    PubMed

    Joo, Hyun; Chavan, Archana G; Fraga, Keith J; Tsai, Jerry

    2015-12-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map.

  3. An Amino Acid Code for Irregular and Mixed Protein Packing

    PubMed Central

    Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

    2015-01-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

  4. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    PubMed Central

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  5. Reversible lysine modification on proteins by using functionalized boronic acids.

    PubMed

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

  6. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  7. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts.

  8. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  9. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    PubMed Central

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael C.

    2014-01-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications. PMID:24959531

  10. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  11. Photo-CIDNP NMR spectroscopy of amino acids and proteins.

    PubMed

    Kuhn, Lars T

    2013-01-01

    Photo-chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance (NMR) phenomenon which, among other things, is exploited to extract information on biomolecular structure via probing solvent-accessibilities of tryptophan (Trp), tyrosine (Tyr), and histidine (His) amino acid side chains both in polypeptides and proteins in solution. The effect, normally triggered by a (laser) light-induced photochemical reaction in situ, yields both positive and/or negative signal enhancements in the resulting NMR spectra which reflect the solvent exposure of these residues both in equilibrium and during structural transformations in "real time". As such, the method can offer - qualitatively and, to a certain extent, quantitatively - residue-specific structural and kinetic information on both the native and, in particular, the non-native states of proteins which, often, is not readily available from more routine NMR techniques. In this review, basic experimental procedures of the photo-CIDNP technique as applied to amino acids and proteins are discussed, recent improvements to the method highlighted, and future perspectives presented. First, the basic principles of the phenomenon based on the theory of the radical pair mechanism (RPM) are outlined. Second, a description of standard photo-CIDNP applications is given and it is shown how the effect can be exploited to extract residue-specific structural information on the conformational space sampled by unfolded or partially folded proteins on their "path" to the natively folded form. Last, recent methodological advances in the field are highlighted, modern applications of photo-CIDNP in the context of biological NMR evaluated, and an outlook into future perspectives of the method is given.

  12. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  13. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  14. Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    SciTech Connect

    Cingolani, Gino Andrews, Dewan; Casjens, Sherwood

    2006-05-01

    The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

  15. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding.

  16. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  17. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  18. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  19. Shedding light on proteins, nucleic acids, cells, humans and fish

    NASA Technical Reports Server (NTRS)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  20. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  1. Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

    PubMed

    Yeh, Fu-Lung; Tung, Luh; Chang, Tien-Hsien

    2016-01-01

    Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

  2. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  3. Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score of three Mediterranean cephalopods.

    PubMed

    Zlatanos, Spiros; Laskaridis, Kostas; Feist, Christian; Sagredos, Angelos

    2006-10-01

    Proximate composition, fatty acid analysis and protein digestibility-corrected amino acid score (PDCAAS) in three commercially important cephalopods of the Mediterranean sea (cuttlefish, octopus and squid) were determined. The results of the proximate analysis showed that these species had very high protein:fat ratios similar to lean beef. Docosahexaenoic, palmitic and eicosipentaenoic acid were the most abundant fatty acids among analyzed species. The amount of n-3 fatty acids was higher than that of saturated, monounsaturated and n-6 fatty acids. Despite the fact that cephalopods contain small amounts of fat they were found quite rich in n-3 fatty acids. Finally, PDCAAS indicated that these organisms had a very good protein quality.

  4. Maternal folic acid supplementation to dams on marginal protein level alters brain fatty acid levels of their adult offspring.

    PubMed

    Rao, Shobha; Joshi, Sadhana; Kale, Anvita; Hegde, Mahabaleshwar; Mahadik, Sahebarao

    2006-05-01

    Studies on fetal programming of adult diseases have highlighted the importance of maternal nutrition during pregnancy. Folic acid and long-chain essential polyunsaturated fatty acids (LC-PUFAs) have independent effects on fetal growth. However, folic acid effects may also involve alteration of LC-PUFA metabolism. Because marginal deficiency of LC-PUFAs during critical periods of brain growth and development is associated with risks for adult diseases, it is highly relevant to investigate how maternal supplementation of such nutrients can alter brain fatty acid levels. We examined the impact of folic acid supplementation, conventionally used in maternal intervention, on brain essential fatty acid levels and plasma corticosterone concentrations in adult offspring at 11 months of age. Pregnant female rats from 4 groups (6 in each) were fed with casein diets either with 18 g protein/100 g diet (control diet) or treatment diets that were marginal in protein (MP), such as 12 g protein/100 g diet supplemented with 8 mg folic acid (FAS/MP), 12 g protein/100 g diet without folic acid (FAD/MP), or 12 g protein/100 g diet (MP) with 2 mg folic acid. Pups were weaned to a standard laboratory diet with 18 g protein/100 g diet. All male adult offspring in the FAS/MP group showed lower docosahexaenoic acid (P<.05) as compared with control adult offspring (6.04+/-2.28 vs 10.33+/-0.86 g/100 g fatty acids) and higher n-6/n-3 ratio (P<.05). Docosahexaenoic acid levels in FAS/MP adult offspring were also lower (P<.05) when compared with the MP group. Plasma corticosterone concentrations were higher (P<.05) in male adult offspring from the FAS/MP group compared with control as well as the MP adult offspring. Results suggest that maternal folic acid supplementation at MP intake decreased brain docosahexaenoic acid levels probably involving corticosterone increase.

  5. Assessing the Chemical Accuracy of Protein Structures via Peptide Acidity

    PubMed Central

    Anderson, Janet S.; Hernández, Griselda; LeMaster, David M.

    2012-01-01

    Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses. PMID:23182463

  6. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein.

  7. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins.

  8. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  9. Defining meal requirements for protein to optimize metabolic roles of amino acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dietary protein provides essential amino acids (EAAs) for the synthesis of new proteins plus an array of other metabolic functions; many of these functions are sensitive to postprandial plasma and intracellular amino acid concentrations. Recent research has focused on amino acids as metabolic signal...

  10. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  11. Exogenous amino acids stimulate net muscle protein synthesis in the elderly.

    PubMed Central

    Volpi, E; Ferrando, A A; Yeckel, C W; Tipton, K D; Wolfe, R R

    1998-01-01

    We have investigated the response of amino acid transport and protein synthesis in healthy elderly individuals (age 71+/-2 yr) to the stimulatory effect of increased amino acid availability. Muscle protein synthesis and breakdown, and amino acid transport were measured in the postabsorptive state and during the intravenous infusion of an amino acid mixture. Muscle-free amino acid kinetics were calculated by means of a three compartment model using data obtained by femoral arterio-venous catheterization and muscle biopsies from the vastus lateralis during the infusion of stable isotope tracers of amino acids. In addition, muscle protein fractional synthetic rate (FSR) was measured. Peripheral amino acid infusion significantly increased amino acid delivery to the leg, amino acid transport, and muscle protein synthesis when measured either with the three compartment model (P < 0.05) or with the traditional precursor-product approach (FSR increased from 0. 0474+/-0.0054 to 0.0940+/-0.0143%/h, P < 0.05). Because protein breakdown did not change during amino acid infusion, a positive net balance of amino acids across the muscle was achieved. We conclude that, although muscle mass is decreased in the elderly, muscle protein anabolism can nonetheless be stimulated by increased amino acid availability. We thus hypothesize that muscle mass could be better maintained with an increased intake of protein or amino acids. PMID:9576765

  12. Alternative splicing of CARMA2/CARD14 transcripts generates protein variants with differential effect on NF-κB activation and endoplasmic reticulum stress-induced cell death.

    PubMed

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-12-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis.

  13. Alternative Splicing of CARMA2/CARD14 Transcripts Generates Protein Variants With Differential Effect on NF-κB Activation and Endoplasmic Reticulum Stress-Induced Cell Death

    PubMed Central

    Scudiero, Ivan; Zotti, Tiziana; Ferravante, Angela; Vessichelli, Mariangela; Vito, Pasquale; Stilo, Romania

    2011-01-01

    The caspase recruitment domain (CARD)-containing proteins CARMA1-3 share high degree of sequence, structure and functional homology. Whereas CARMA1 and CARMA3 have been identified as crucial components of signal transduction pathways that lead to activation of NF-κB transcription factor, little is known about the function of CARMA2. Here we report the identification of two splice variants of CARMA2. One transcript, named CARMA2short (CARMA2sh), is predicted to encode for a CARMA2 polypeptide containing the CARD, coiled coil, and a PDZ domains, but lacking the SH3 and the GuK domains. The second variant, CARMA2cardless (CARMA2cl), encodes for a polypeptide lacking the CARD domain and containing only a portion of the coiled coil domain and a linker region. Expression analysis confirmed the presence of the CARMA2 alternatively spliced transcripts in both human cell lines and tissues. Fluorescence microscopy data show that both splice variants localize in the cytosol. Biochemical experiments indicate that CARMA2sh interacts with TRAF2 and activates NF-κB in a TRAF2-dependent manner. Finally, CARMA2sh variant protects cells from apoptosis induced by different stimuli. Taken together, these results demonstrate that multiple transcripts encoding several CARMA2 isoforms exist in vivo and regulate NF-κB activation and apoptosis. J. Cell. Physiol. 226: 3121–3131, 2011. © 2011 Wiley Periodicals, Inc. PMID:21302310

  14. Toucan protein is essential for the assembly of syncytial mitotic spindles in Drosophila melanogaster.

    PubMed

    Debec, A; Grammont, M; Berson, G; Dastugue, B; Sullivan, W; Couderc, J L

    2001-12-01

    The toc gene of Drosophila melanogaster encodes a 235-kD polypeptide with a coiled-coil domain, which is highly expressed during oogenesis (Grammont et al., 1997, 2000). We now report the localization of the Toucan protein during early embryonic development. The Toucan protein is present only during the syncytial stages and is associated with the nuclear envelope and the cytoskeletal structures of the syncytial embryo. In anaphase A, Toucan is concentrated at the spindle poles near the minus end of microtubules. This microtubule association is very dynamic during the nuclear cell cycle. Mutant embryos lacking the Toucan protein are blocked in a metaphase-like state. They display abnormal and nonfunctional spindles, characterized by broad poles, detachment of the centrosomes, and failure of migration of the chromosomes. These results strongly suggest that Toucan represents a factor essential for the assembly and the function of the syncytial mitotic spindles.

  15. Production in Pichia pastoris of protein-based polymers with small heterodimer-forming blocks.

    PubMed

    Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

    2016-05-01

    Some combinations of leucine zipper peptides are capable of forming α-helical heterodimeric coiled coils with very high affinity. These can be used as physical cross-linkers in the design of protein-based polymers that form supramolecular structures, for example hydrogels, upon mixing solutions containing the complementary blocks. Such two-component physical networks are of interest for many applications in biomedicine, pharmaceutics, and diagnostics. This article describes the efficient secretory production of A and B type leucine zipper peptides fused to protein-based polymers in Pichia pastoris. By adjusting the fermentation conditions, we were able to significantly reduce undesirable proteolytic degradation. The formation of A-B heterodimers in mixtures of the purified products was confirmed by size exclusion chromatography. Our results demonstrate that protein-based polymers incorporating functional heterodimer-forming blocks can be produced with P. pastoris in sufficient quantities for use in future supramolecular self-assembly studies and in various applications.

  16. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  17. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  18. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  19. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  20. Interfacial inhibitors of protein-nucleic acid interactions.

    PubMed

    Pommier, Yves; Marchand, Christophe

    2005-07-01

    This essay develops the paradigm of "Interfacial Inhibitors" (Pommier and Cherfils, TiPS, 2005, 28: 136) for inhibitory drugs beside orthosteric (competitive or non-competitive) and allosteric inhibitors. Interfacial inhibitors bind with high selectivity to a binding site involving two or more macromolecules within macromolecular complexes undergoing conformational changes. Interfacial binding traps (generally reversibly) a transition state of the complex, resulting in kinetic inactivation. The exemplary case of interfacial inhibitor of protein-DNA interface is camptothecin and its clinical derivatives. We will also provide examples generalizing the interfacial inhibitor concept to inhibitors of topoisomerase II (anthracyclines, ellipticines, epipodophyllotoxins), gyrase (quinolones, ciprofloxacin, norfloxacin), RNA polymerases (alpha-amanitin and actinomycin D), and ribosomes (antibiotics such as streptomycin, hygromycin B, tetracycline, kirromycin, fusidic acid, thiostrepton, and possibly cycloheximide). We discuss the implications of the interfacial inhibitor concept for drug discovery.

  1. Parsing the life-shortening effects of dietary protein: effects of individual amino acids.

    PubMed

    Arganda, Sara; Bouchebti, Sofia; Bazazi, Sepideh; Le Hesran, Sophie; Puga, Camille; Latil, Gérard; Simpson, Stephen J; Dussutour, Audrey

    2017-01-11

    High-protein diets shorten lifespan in many organisms. Is it because protein digestion is energetically costly or because the final products (the amino acids) are harmful? To answer this question while circumventing the life-history trade-off between reproduction and longevity, we fed sterile ant workers on diets based on whole proteins or free amino acids. We found that (i) free amino acids shortened lifespan even more than proteins; (ii) the higher the amino acid-to-carbohydrate ratio, the shorter ants lived and the lower their lipid reserves; (iii) for the same amino acid-to-carbohydrate ratio, ants eating free amino acids had more lipid reserves than those eating whole proteins; and (iv) on whole protein diets, ants seem to regulate food intake by prioritizing sugar, while on free amino acid diets, they seem to prioritize amino acids. To test the effect of the amino acid profile, we tested diets containing proportions of each amino acid that matched the ant's exome; surprisingly, longevity was unaffected by this change. We further tested diets with all amino acids under-represented except one, finding that methionine, serine, threonine and phenylalanine are especially harmful. All together, our results show certain amino acids are key elements behind the high-protein diet reduction in lifespan.

  2. Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.

    PubMed

    Torshin, I Y

    2001-04-01

    Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

  3. Thermodynamics of the interaction of globular proteins with powdered stearic acid in acid pH.

    PubMed

    Mitra, Atanu; Chattoraj, D K; Chakraborty, P

    2006-06-01

    Adsorption isotherms of different globular proteins and gelatin on strearic acid particles have been studied as a function of biopolymer concentration, ionic strength of the medium, and temperature. The effect of neutral salts including CaCl2, Na3PO4, and urea on the adsorption isotherms has been also investigated. It is observed that the extent of adsorption (Gamma2(1)) increases in two steps with the increase of biopolymer concentration (C2) in the bulk. Gamma2(1) increases with an increase of C2 until a steady maximum value Gamma2(m) is reached at a critical concentration C2(m). After initial saturation, Gamma2(1) again increases from Gamma2(m) without reaching any limiting value due to the surface aggregation of the protein. The values of the standard free energy change for adsorption have been calculated on the basis of the Gibbs equation. The standard entropy and enthalpy changes are also calculated.

  4. Determination of the amount of protein and amino acids extracted from the microbial protein (SCP) of lignocellulosic wastes.

    PubMed

    Ahmadi, A R; Ghoorchian, H; Hajihosaini, R; Khanifar, J

    2010-04-15

    With the increasing world population, the use of lignocellulosic wastes for production of microbial protein as animal feed becomes a necessity of our time. In order to verify the most productive protein, the amount of protein and amino acid extracted from Single Cell Protein (SCP) needs to be determined by an effective method. In this study Microbial protein was produced by treatment of wheat straw with Pleurotus florida; with heat at 100 degrees C and NaOH 2% as substrate by solid state fermentation. Concentration of protein was 62.8% per 100 g of dried microbial protein. Then the extracted protein hydrolyzed with HCl 6 Normal for 48 h under 110 degrees C temperature condition. Then the amino acids analyzed by using A-200 Amino Nova analyzer. The results of this study indicated that the ratio of essential amino acids to total amino acids was 65.6%. The concentration of essnyial amino acids were: Lysine = 9.5, histidine = 19.8, threonine = 0.6, valine = 6.6, methionine = 2.1, isoleucine = 7.3, leucine = 6.8, phenylalanine = 4.3 and arginine = 8.3 g/100 g of extracted protein that indicated the obtained microbial protein can be a good or suitable substitute in the food program of animal feed.

  5. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  6. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

  7. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  8. PROTEIN METABOLISM IN REGENERATING WOUND TISSUE: FUNCTION OF THE SULFUR AMINO ACIDS.

    DTIC Science & Technology

    PROTEINS, *TISSUES(BIOLOGY), METABOLISM, TISSUES(BIOLOGY), REGENERATION(ENGINEERING), WOUNDS AND INJURIES, TISSUES(BIOLOGY), TRACER STUDIES, METHIONINE, COLLAGEN, TYROSINE, BIOSYNTHESIS, AMINO ACIDS .

  9. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  10. Amino acid composition of Lagenaria siceraria seed flour and protein fractions.

    PubMed

    Ogunbusola, Moriyike Esther; Fagbemi, Tayo Nathaniel; Osundahunsi, Oluwatooyin Faramade

    2010-12-01

    Defatted seed flours of Lagenaria siceraria (calabash