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Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein 总被引:3,自引:0,他引:3
Seog DH 《Bioscience, biotechnology, and biochemistry》2004,68(8):1808-1810
To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites. 相似文献
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In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ~20% of the total cellular protein. 相似文献
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Kamei A Hauser PS Beckstead JA Weers PM Ryan RO 《Protein expression and purification》2012,83(2):113-116
Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I(Milano)(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I(Milano). The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size. 相似文献
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Lusetti SL Voloshin ON Inman RB Camerini-Otero RD Cox MM 《The Journal of biological chemistry》2004,279(29):30037-30046
When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response. 相似文献
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In living systems, the chemical space and functional repertoire of proteins are dramatically expanded through the post-translational modification (PTM) of various amino acid residues. These modifications frequently trigger unique protein–protein interactions (PPIs) – for example with reader proteins that directly bind the modified amino acid residue – which leads to downstream functional outcomes. The modification of a protein can also perturb its PPI network indirectly, for example, through altering its conformation or subcellular localization. Uncovering the network of unique PTM-triggered PPIs is essential to fully understand the roles of an ever-expanding list of PTMs in our biology. In this review, we discuss established strategies and current challenges associated with this endeavor. 相似文献
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One of the major goals of molecular and evolutionary biology is to understand the functions of proteins by extracting functional
information from protein sequences, structures and interactions. In this review, we summarize the repertoire of methods currently
being applied and report recent progress in the field of in silico annotation of protein function based on the accumulation
of vast amounts of sequence and structure data. In particular, we emphasize the newly developed structure-based methods, which
are able to identify locally structural motifs and reveal their relationship with protein functions. These methods include
computational tools to identify the structural motifs and reveal the strong relationship between these pre-computed local
structures and protein functions. We also discuss remaining problems and possible directions for this exciting and challenging
area. 相似文献
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Huang PS Love JJ Mayo SL 《Protein science : a publication of the Protein Society》2007,16(12):2770-2774
As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid beta1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest ( approximately 300 microM), 2D-[(1)H,(15)N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation. 相似文献
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Colas P 《Current opinion in chemical biology》2000,4(1):54-59
The design and use of combinatorial protein libraries has become a fast moving field in molecular biology. Different experimental systems supporting various selection schemes are now available. The latest breakthroughs include evolutionary experiments to improve existing binding surfaces, selections of homodimerizing peptides, the use of peptide aptamers to disrupt protein interactions inside living cells, and functional selections of aptamers to probe regulatory networks. 相似文献
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Tonoplast-bound protein kinase phosphorylates tonoplast intrinsic protein 总被引:14,自引:1,他引:13
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Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. 相似文献
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Mammalian CHORD-containing protein 1 is a novel heat shock protein 90-interacting protein 总被引:4,自引:0,他引:4
With two tandem repeated cysteine- and histidine-rich domains (designated as CHORD), CHORD-containing proteins (CHPs) are a novel family of highly conserved proteins that play important roles in plant disease resistance and animal development. Through interacting with suppressor of the G2 allele of Skp1 (SGT1) and Hsp90, plant CHORD-containing protein RAR1 (required for Mla resistance 1) plays a critical role in disease resistance mediated by multiple R genes. Yet, the physiological function of vertebrate CHORD-containing protein-1 (Chp-1) has been poorly investigated. In this study, we provide the first biochemical evidence demonstrating that mammalian Chp-1 is a novel Hsp90-interacting protein. Mammalian Chp-1 contains two CHORD domains (I and II) and one CS domain (a domain shared by CHORD-containing proteins and SGT1). With sequence and structural similarity to Hsp90 co-chaperones p23 and SGT1, Chp-1 binds to the ATPase domain of Hsp90, but the biochemical property of the interaction is unique. The Chp-1-Hsp90 interaction is independent of ATP and ATPase-coupled conformational change of Hsp90, a feature that distinguishes Chp-1 from p23. Furthermore, it appears that multiple domains of Chp-1 are required for stable Chp-1-Hsp90 interaction. Unlike SGT1 whose CS domain is sufficient for Hsp90 binding, the CS domain of Chp-1 is essential but not sufficient for Hsp90 binding. While the CHORD-I domain of Chp-1 is dispensable for Hsp90 binding, the CHORD-II domain and the linker region are essential. Interestingly, the CHORD-I domain of plant RAR1 protein is solely responsible for Hsp90 binding. The unique Chp-1-Hsp90 interaction may be indicative of a distinct biological activity of Chp-1 and functional diversification of CHORD-containing proteins during evolution. 相似文献
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Death-associated protein kinase (DAPK) is a pro-apoptotic, calcium/calmodulin-regulated protein kinase that is a drug discovery target for neurodegenerative disorders. Despite the potential profound physiological role of DAPK in neuronal function and pathophysiology, the endogenous substrate(s) of this kinase and the mechanisms via which DAPK elicits its biological action remain largely unknown. We report here that the mammalian 40S ribosomal protein S6 is a DAPK substrate. Results from immunoprecipitation experiments are consistent with endogenous DAPK being associated with endogenous S6 in rat brain. When S6 is a component of the 40S ribosomal subunit complex, DAPK selectively phosphorylates it at serine 235, one of the five sites in S6 that are phosphorylated by the S6 kinase family of proteins. The amino acid sequence flanking serine 235 matches the established pattern for DAPK peptide and protein substrates. Kinetic analyses using purified 40S subunits revealed a K(m) value of 9 microM, consistent with S6 being a potential physiological substrate of DAPK. This enzyme-substrate relationship has functional significance. DAPK suppresses translation in rabbit reticulocyte lysate, and treatment of neuroblastoma cells with a stimulator of DAPK reduces protein synthesis. In both cases, suppression of translation correlates with increased phosphorylation of S6 at serine 235. These results demonstrate that DAPK is a S6 kinase and provide evidence for a novel role of DAPK in the regulation of translation. 相似文献
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ABSTRACT: Co-evolving positions within protein sequences have been used as spatial constraints to develop a computational approach for modeling membrane protein structures. 相似文献
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The high-throughput structure determination pipelines developed by structural genomics programs offer a unique opportunity
for data mining. One important question is how protein properties derived from a primary sequence correlate with the protein’s
propensity to yield X-ray quality crystals (crystallizability) and 3D X-ray structures. A set of protein properties were computed
for over 1,300 proteins that expressed well but were insoluble, and for ~720 unique proteins that resulted in X-ray structures.
The correlation of the protein’s iso-electric point and grand average hydropathy (GRAVY) with crystallizability was analyzed
for full length and domain constructs of protein targets. In a second step, several additional properties that can be calculated
from the protein sequence were added and evaluated. Using statistical analyses we have identified a set of the attributes
correlating with a protein’s propensity to crystallize and implemented a Support Vector Machine (SVM) classifier based on
these. We have created applications to analyze and provide optimal boundary information for query sequences and to visualize
the data. These tools are available via the web site . 相似文献
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Improvement of protein stability in protein microarrays 总被引:1,自引:0,他引:1
Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml–1 to 5 g ml–1 by fluorescence analysis. 相似文献
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Chudakov DM Verkhusha VV Staroverov DB Souslova EA Lukyanov S Lukyanov KA 《Nature biotechnology》2004,22(11):1435-1439
In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications. 相似文献
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Patterns of protein protein interactions in salt solutions and implications for protein crystallization 总被引:1,自引:0,他引:1
Dumetz AC Snellinger-O'brien AM Kaler EW Lenhoff AM 《Protein science : a publication of the Protein Society》2007,16(9):1867-1877
The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization. 相似文献