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1.
Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed. 相似文献
2.
Despite the clear importance of the left-handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix-forming propensities measured for all residues, except tyrosine and tryptophan, in a proline-based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix-forming propensity scale will prove useful in future studies of the conformational properties of proline-rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states. 相似文献
3.
Jose Manuel Martin‐Garcia Irene Luque Javier Ruiz‐Sanz Ana Camara‐Artigas 《Acta Crystallographica. Section D, Structural Biology》2012,68(8):1030-1040
The hepatitis C virus nonstructural 5A (NS5A) protein is a large zinc‐binding phosphoprotein that plays an important role in viral RNA replication and is involved in altering signal transduction pathways in the host cell. This protein interacts with Fyn tyrosine kinase in vivo and regulates its kinase activity. The 1.5 Å resolution crystal structure of a complex between the SH3 domain of the Fyn tyrosine kinase and the C‐terminal proline‐rich motif of the NS5A‐derived peptide APPIPPPRRKR has been solved. Crystals were obtained in the presence of ZnCl2 and belonged to the tetragonal space group P41212. The asymmetric unit is composed of four SH3 domains and two NS5A peptide molecules; only three of the domain molecules contain a bound peptide, while the fourth molecule seems to correspond to a free form of the domain. Additionally, two of the SH3 domains are bound to the same peptide chain and form a ternary complex. The proline‐rich motif present in the NS5A protein seems to be important for RNA replication and virus assembly, and the promiscuous interaction of the Fyn SH3 domain with the NS5A C‐terminal proline‐rich peptide found in this crystallographic structure may be important in the virus infection cycle. 相似文献
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5.
Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system. 相似文献
6.
Development of sequence-based methods for predicting putative interfacial residues is an extremely important task in modeling 3D structures of protein–protein complexes. In the present paper we used non-gapped sequence segments to predict both interacting and interfacial residues. We demonstrated that continuous sequence segments do occur at the protein–protein interfaces and showed that continuous interacting interfacial segments (CIIS) of length nine are presented on average, in 37% of the complexes in our dataset. Our results indicate that CIIS consist mostly of interacting strands and/or loops, while the CIIS involving the helixes are scarce. We performed scoring of CIIS using four different scoring mechanisms and found that scores of CIIS differ significantly from the scores calculated for random stretches of residues. We argue that such statistical difference inferred thought the corresponding Z-scores could be used for detecting putative interfacial residue segments without using any structural information. This hypothesis was tested on our dataset and benchmarking resulted to 10–60% prediction accuracy depending on type of benchmarking and scoring scheme used in calculations. Such predictions that do not depend on the availability of the 3D structures of monomers can be quite valuable in modeling 3D structures of obligatory complexes, for which structures of separated monomers do not exist. 相似文献
7.
Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture. 相似文献
8.
The original vectors of the bacterial two-hybrid technique developed by Karimova et al. in 1998 did not enable detection of the recombinant proteins. Here, we propose two methods resolving this problem, either using new plasmids containing the Flag epitope, or using a trick to detect the T18 domain of adenylate cyclase. Furthermore, we describe a set of vectors for TAP, CBP or 6-histidine tagging that possess the same cloning site as our two-hybrid vectors. 相似文献
9.
Chiara Rubini Paolo Ruzza Mark R. Spaller Giuliano Siligardi Rohanah Hussain D. Gomika Udugamasooriya Massimo Bellanda Stefano Mammi Andrea Borgogno Andrea Calderan Luca Cesaro Anna M. Brunati Arianna Donella‐Deana 《Peptide Science》2010,94(3):298-306
Cortactin is a ubiquitous actin‐binding protein that regulates various aspects of cell dynamics and is implicated in the pathogenesis of human neoplasia. The sequence of cortactin contains a number of signaling motifs and an SH3 domain at the C‐terminus, which mediates the interaction of the protein with several partners, including Shank2. A recombinant protein, comprising the murine cortactin SH3 domain fused to GST (GST‐SH3m‐cort), was prepared and used to assess the domain‐binding affinity of potential peptide‐ligands reproducing the proline‐rich regions of human HPK1 and Shank2 proteins. The key residues involved in the SH3m‐cort domain recognition were identified by three different approaches: non‐immobilized ligand interaction assay by circular dichroism, isothermal titration calorimetry, and nuclear magnetic resonance. Our results show that the classical PxxPxK class II binding motif is not sufficient to mediate the interaction with GST‐SH3m‐cort, an event that depends on the presence of additional basic residues located at either the N‐ or the C‐terminus of the PxxPxK motif. Especially effective in promoting the peptide binding is a Lys residue at the ‐5 position, a determinant present in both P2 (HPK1 394‐403) and S1 (Shank2 1168‐1189) peptides. GST‐SH3m‐cort exhibits the highest affinity toward peptide S1, which contains additional Lys residues at the ‐3, ‐5, and ‐7 positions, indicating that the optimal consensus motif may be KPPxPxKxKxK. These results are supported by the in silico models of SH3m‐cort complexed with P2 or S1, which highlight the domain residues that interact with the recognition determinants of the peptide‐ligand and cooperate in binding stabilization. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 298–306, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
10.
Paolo Ruzza Arianna Donella‐Deana Andrea Calderan Annamaria Brunati Maria Lina Massimino Stefano Elardo Alessio Mattiazzo Lorenzo A. Pinna Gianfranco Borin 《Peptide Science》2001,60(4):290-306
With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src‐like tyrosine kinases, we synthesized a tyrosyl‐phospho decapeptide, corresponding to the sequence 392‐401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src‐like kinases c‐Fgr or Lyn. This phospho‐peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane‐mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c‐Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N‐terminal Antennapedia fragment only slightly affects the binding of the phospho‐HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho‐HS1 peptide and its chimeric derivative greatly affect either the SH2‐mediated recruitment of the c‐Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src‐like kinase c‐Fgr. The ability of the chimeric phospho‐peptide to enter cells has been demonstrated by confocal microscopy analysis. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 290–306, 2001 相似文献
11.
Marina Gobbo Laura Biondi Fernando Filira Raniero Rocchi 《Journal of peptide science》2006,12(2):132-139
Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane. 相似文献
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Ana Camara-Artigas Emilia Ortiz-Salmeron Montserrrat Andujar-Snchez Julio Bacarizo Jose Manuel Martin-Garcia 《Acta Crystallographica. Section F, Structural Biology Communications》2016,72(9):707-712
Interactions of proline‐rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein–protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high‐affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation–π and salt‐bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family. 相似文献
14.
Paolo Ruzza Giuliano Siligardi Arianna Donella-Deana Andrea Calderan Rohanah Hussain Chiara Rubini Luca Cesaro Alessio Osler Andrea Guiotto Lorenzo A Pinna Gianfranco Borin 《Journal of peptide science》2006,12(7):462-471
Eukaryotic signal transduction involves the assembly of transient protein-protein complexes mediated by modular interaction domains. Specific Pro-rich sequences with the consensus core motif PxxP adopt the PPII helix conformation upon binding to SH3 domains. For short Pro-rich peptides, little or no ordered secondary structure is usually observed before binding interactions. The association of a Pro-rich peptide with the SH3 domain involves unfavorable binding entropy due to the loss of rotational freedom on forming the PPII helix. With the aim of stabilizing the PPII helix conformation in the Pro-rich HPK1 decapeptide PPPLPPKPKF (P2), a series of P2 analogues was prepared, in which specific Pro positions were alternatively occupied by 4(S)- or 4(R)-4-fluoro-L-proline. The interactions of these peptides with the SH3 domain of the HPK1-binding partner HS1 were quantitatively analyzed by the NILIA-CD approach. A CD thermal analysis of the P2 analogues was performed to assess their propensity to adopt the PPII helix conformation. Contrary to our expectations, the K(d) values of the analogues were lower than that of the parent peptide P2. These results clearly show that the induction of a stable PPII helix conformation in short Pro-rich peptides is not sufficient to increase their affinity toward the SH3 domain and that the effect of 4-fluoroproline strongly depends on the position of this residue in the sequence and the chirality of the substituent in the pyrrolidine ring. 相似文献
15.
Benedetta Righino Davide Pirolli Giorgia Radicioni Valeria Marzano Renato Longhi Alessandro Arcovito Maria Teresa Sanna Maria Cristina De Rosa Serena Paoluzi Gianni Cesareni Irene Messana Massimo Castagnola Alberto Vitali 《Peptide Science》2016,106(5):714-725
Human saliva contains hundreds of small proline‐rich peptides originated by the proteolytic cleavage of the salivary basic Proline‐Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti‐HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline‐II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c‐Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714–725, 2016. 相似文献
16.
Ojennus DD Lehto SE Wuttke DS 《Protein science : a publication of the Protein Society》2003,12(1):44-55
Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9-kD and 5-kD peptide fragments formed by limited proteolytic digestion of the N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3'-kinase fold into an active native-like structure on interaction with one another. The corresponding 5-kD fragment of the homologous Src protein, however, was not capable of structurally complementing the p85 9-kD fragment, indicating that fragment complementation among these SH2 domains is sensitive to the sequence differences between the Src and p85 domains. Partial complementation and folding activity could be recovered with hybrid sequences of these SH2 domains. Complete alanine scanning of the 5-kD p85 fragment was used to identify the sequence recognition elements required for complex formation. The alanine substitutions in the p85 5-kD fragment that abolished binding affinity with the cognate 9-kD fragment correlate well with highly conserved residues among SH2 domains that are either integrally involved in core packing or found at the interface between fragments. Surprisingly, however, mutation of a nonconserved surface-exposed aspartic acid to alanine was found to have a significant effect on complementation. A single additional mutation of arginine to aspartic acid allowed for recovery of native structure and increased the thermal stability of the designed Src-p85 chimera by 18 degrees C. This modification appears to relieve an unfavorable surface electrostatic interaction, demonstrating the importance of surface charge interactions in protein stability. 相似文献
17.
To assess the role of 14-3-3 proteins in the magnesium-dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14-3-3s by coimmunoprecipitation and gel filtration. The stability of the 14-3-3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14-3-3s per se does not inhibit NR. Magnesium inhibited 14-3-3-bound NR much more strongly than 14-3-3-free NR. 14-3-3s possibly reinforce NR inhibition by magnesium. 相似文献
18.
The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline‐rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs‐bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide‐containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, β‐d ‐glucosyl‐formaecin I and β‐d ‐glucosyl‐drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that β‐d ‐maltosyl‐formaecin I and β‐d ‐maltosyl‐drosocin were more potent than that of respective β‐d ‐Glc‐analog, β‐d ‐cellobiosyl‐analog and β‐d ‐lactosyl‐analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline‐rich class of antibacterial peptides. The comparative–quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline‐rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
19.
Stefanie Fritsche Daniel Knappe Nicole Berthold Heiner von Buttlar Ralf Hoffmann Gottfried Alber 《Journal of peptide science》2012,18(10):599-608
Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
20.
Mikula M Dzwonek A Karczmarski J Rubel T Dadlez M Wyrwicz LS Bomsztyk K Ostrowski J 《Proteomics》2006,6(8):2395-2406
The heterogeneous nuclear ribonucleoprotein K is an ancient RNA/DNA-binding protein that is involved in multiple processes that compose gene expression. The pleiotropic action of K protein reflects its ability to interact with different classes of factors, interactions that are regulated by extracellular signals. We used affinity purification and MS to better define the repertoire of K protein partners. We identified a large number of new K protein partners, some typically found in subcellular compartments, such as plasma membrane, where K protein has not previously been seen. Electron microscopy showed K protein in the nucleus, cytoplasm, mitochondria, and in vicinity of plasma membrane. These observations greatly expanded the view of the landscape of K protein-protein interaction and provide new opportunities to explore signal transduction and gene expression in several subcellular compartments. 相似文献