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1.
根据PDB提供的PrPC的原子坐标,利用MSMS程序,对PrPC氨基酸残基溶剂可及表面积进行了计算和分析.结果表明:(1) PrPC氨基酸残基可及性具有总体一致性特点;(2) PrPC蛋白质序列中非保守残基与种间屏障有一定关系;(3) 在PrPC向PrP Sc转变过程中,由于蛋白质X的结合,PrPC可能会出现一定的构象变化,这种变化利于PrPC向PrPSc发生转变. 相似文献
2.
F C Purves A D Deana F Marchiori D P Leader L A Pinna 《Biochimica et biophysica acta》1986,889(2):208-215
Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with alpha-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simplex virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different. 相似文献
3.
Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity 总被引:6,自引:0,他引:6
I Clark-Lewis A F Lopez L B To M A Vadas J W Schrader L E Hood S B Kent 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(3):881-889
Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected. 相似文献
4.
Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein 总被引:3,自引:0,他引:3
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Takemasa Sakaguchi Tsuneo Uchiyama Cheng Huang Noriko Fukuhara Katsuhiro Kiyotani Yoshiyuki Nagai Tetsuya Yoshida 《Journal of virology》2002,76(4):1682-1690
The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C(83)S, SeV M-C(106)S, and SeV M-C(295)S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C(83)S and SeV M-C(106)S had smaller virus particles than did the wild-type SeV, whereas SeV M-C(295)S had larger and heterogeneously sized particles. Furthermore, SeV M-C(106)S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle. 相似文献
5.
Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3 总被引:9,自引:0,他引:9
R K Kaul S N Murthy A G Reddy T L Steck H Kohler 《The Journal of biological chemistry》1983,258(13):7981-7990
We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites. 相似文献
6.
Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein. 总被引:20,自引:9,他引:11
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The Tat transactivator protein of human immunodeficiency virus type 1 contains a highly conserved cysteine-rich region, containing seven cysteines from residues 22 through 37. To investigate the importance of noncysteine residues in this region of the Tat protein, we have carried out a mutational analysis, in most cases substituting a single alanine for the wild-type noncysteine residue. Alanine substitution of residue 23, 24, 46, or 47 had no effect on Tat activity in plasmid transfection assays. In contrast, alanine substitutions of all eight noncysteines analyzed, from residues 26 through 41, significantly reduced the activity of the Tat protein, in some cases as drastically as mutations in cysteine residues. The results demonstrate that the precise sequence of the cysteine-rich region is crucial for a fully functional Tat protein. 相似文献
7.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,889(2):208-215
Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with α-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simples virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different. 相似文献
8.
The complete amino acids sequence of human complex-forming glycoprotein heterogeneous in charge (protein HC) 总被引:1,自引:0,他引:1
C Lopez A Grubb F Soriano E Mendez 《Biochemical and biophysical research communications》1981,103(3):919-925
The complete amino acid sequence of human protein HC isolated from the urine of a single individual (JL) was determined. The polypeptide chain contained 181 residues, had a calculated molecular weight of 20,435 and contained 3 cysteine residues at positions 34, 70 and 167. An intrachain disulfide bridge was formed by residues 70 and 167. No variation of the amino acid sequence of protein HC was found and can therefore not explain the charge heterogeneity of protein HC in a given individual. The amino acid sequence of protein HC was almost identical to the one reported for human α1-microglobulin but contained 14 additional residues. 相似文献
9.
Further definition of the substrate specificity of the alpha-herpesvirus protein kinase and comparison with protein kinases A and C 总被引:6,自引:0,他引:6
D P Leader A D Deana F Marchiori F C Purves L A Pinna 《Biochimica et biophysica acta》1991,1091(3):426-431
The pseudorabies virus protein kinase prefers model substrates containing arginyl residues on the amino-terminal side of a target seryl or threonyl residue. We have defined this substrate specificity more precisely in experiments using a new series of synthetic model peptides. When the number of arginyl residues was varied from two to four in substrates of the type RnASVA it was found that peptides with four arginyl residues constituted the best substrates, although the most marked decrease in Km was seen on increasing the number of arginyl residues from two to three. The effect of varying the number of 'spacer' alanyl residues from zero to three was investigated in peptides of the type R4AmSVA, and the peptide with one alanyl residue was found to be the best substrate, making R4X the optimal amino-terminal environment for this enzyme. A similar substrate specificity was observed with the herpes simplex type 1 protein kinase. Protein kinase C was found to have a quite similar substrate preference to the viral enzyme as far as the number and position of the amino-terminal basic residues was concerned; but, unlike the viral protein kinase, it also requires carboxy-terminal basic residues in optimal peptide substrates, and can tolerate the substitution of lysyl for arginyl residues. The cyclic AMP-dependent protein kinase, like the viral enzyme, had favourable kinetic constants for this series of peptides, but differed from the latter in being able to catalyze the phosphorylation of the peptides with two to four arginyl residues with similar efficiency. Studies with the protein, clupeine Y1, as substrate indicated that the pseudorabies virus protein kinase can tolerate arginyl residues on the carboxyl-terminal side of its target residue when there are suitable amino-terminal arginyl determinants. In this respect the virus protein kinase resembled protein kinase C but differed from the cyclic AMP-dependent protein kinase which cannot tolerate such carboxyl-terminal basic residues. The relationship of substrate specificity with model peptides to the ability of the pseudorabies virus protein kinase to phosphorylate proteins in vitro and in vivo is discussed. 相似文献
10.
11.
用各种化学试剂修饰红花菜豆(Phaseoluscoccineusvarrubronanus,Berry)凝集素(简称PCL)分子,测定与其活性相关的氨基酸残基.经NBS修饰表明PCL具有8个Trp残基,其中4个暴露于分子表面,此4个Trp残基被修饰后,PCL的凝血活性完全丧失.比较PCL修饰前后的CD光谱表明修饰不改变其二级结构。修饰Tyr,Arg,His残基和游离氨基及羧基不影响PCL的血凝活性.巯基也不是血凝活性所必需,但是PCL分子中的二硫键被还原,或被CNBr分解为两个片断则使蛋白质丧失血凝活性,提示分子的完整结构对PCL的血凝活力是重要的 相似文献
12.
Transferring the biological function of one protein to another is a key issue in understanding the structure and function relationship of proteins. We have developed a strategy for grafting protein-protein interaction epitopes. As a first step, residues at the interface of the ligand protein which strongly interact with the receptor protein were identified. Then protein scaffolds were docked onto receptor protein based on geometric complementarity. Only high docking score matches were saved. For each saved match, the scaffold protein was accepted if it had suitable positions for grafting key interaction residues of the ligand protein. These candidate residues were mutated to corresponding residues in the ligand protein at each relevant position and the mutated scaffold protein was co-minimized with receptor protein. Finally, the minimized complexes were evaluated by a scoring function deduced from statistical analysis of rigid binding data sets. As a test case, the binding epitope of barstar, the inhibitor of barnase, was grafted onto smaller proteins. Pheromone Er-1 (PDB entry 1erc) has been found to be a good scaffold. The calculated binding free energy for mutated Pheromone Er-1 is equivalent to that of barstar. 相似文献
13.
Binding sites of rat liver 5S RNA to ribosomal protein L5 总被引:2,自引:0,他引:2
The ribonucleoprotein complex consisting of 5S RNA and the protein L5 was prepared from the large subunit of rat liver ribosomes. The RNA in the complex was digested in situ with RNase A or RNase T1. The RNase-resistant RNA fragments bound to the protein were recovered and purified by 2D-PAGE, and their nucleotide sequences were determined in order to elucidate the binding sites of the RNA to the protein. The results showed that the fragments had arisen from the 5'-end region (residues 1-21), from the second hairpin loop (residues 77-102) and from the 3'-end region (residues 106-120). Harsher digestion trimmed these fragments to shorter fragments. It was concluded that the minimal interactive sequences of 5S RNA to the protein L5 were residues 13-21, residues 85-102, and residues 106-114. A part of the first hairpin loop, residues 41-52, was also suspected to interact with the protein. These protein-binding sites of rat liver 5S RNA were compared with those of Escherichia coli, Halobacterium cutirubrum and yeast, and their probable conservation from eubacteria to eukaryotes is discussed. 相似文献
14.
Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with the corresponding protein from strain 198 总被引:8,自引:0,他引:8
The amino acid sequence of pilin protein from Bacteroides nodosus strain 216 was determined. The protein had a calculated molecular weight of 15962 and contained the same number of amino acid residues (151) as the pilin from the previously sequenced strain 198. The sequence of the first 44 residues was common to both strains, including the unusual amino-terminal amino acid, N-methylphenylalanine. Of the remaining 107 residues, 37% of them differed between the two strains. Comparison of hydrophilicity profiles constructed from the sequence data indicated that a conserved region around residues 71-72 was probably the site of an antigenic determinant. 相似文献
15.
Alsaleh K Delavalle PY Pillez A Duverlie G Descamps V Rouillé Y Dubuisson J Wychowski C 《Journal of virology》2010,84(24):12515-12528
A major function of the hepatitis C virus (HCV) core protein is the interaction with genomic RNA to form the nucleocapsid, an essential component of the virus particle. Analyses to identify basic amino acid residues of HCV core protein, important for capsid assembly, were initially performed with a cell-free system, which did not indicate the importance of these residues for HCV infectivity. The development of a cell culture system for HCV (HCVcc) allows a more precise analysis of these core protein amino acids during the HCV life cycle. In the present study, we used a mutational analysis in the context of the HCVcc system to determine the role of the basic amino acid residues of the core protein in HCV infectivity. We focused our analysis on basic residues located in two clusters (cluster 1, amino acids [aa]6 to 23; cluster 2, aa 39 to 62) within the N-terminal 62 amino acids of the HCV core protein. Our data indicate that basic residues of the first cluster have little impact on replication and are dispensable for infectivity. Furthermore, only four basic amino acids residues of the second cluster (R50, K51, R59, and R62) were essential for the production of infectious viral particles. Mutation of these residues did not interfere with core protein subcellular localization, core protein-RNA interaction, or core protein oligomerization. Moreover, these mutations had no effect on core protein envelopment by intracellular membranes. Together, these data indicate that R50, K51, R59, and R62 residues play a major role in the formation of infectious viral particles at a post-nucleocapsid assembly step. 相似文献
16.
Ito K Sugawara T Koizumi A Nakajima K Shimizu-Ibuka A Shiroishi M Asada H Yurugi-Kobayashi T Shimamura T Asakura T Misaka T Iwata S Kobayashi T Abe K 《Biotechnology letters》2011,33(1):103-107
PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression. 相似文献
17.
The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49. 相似文献
18.
Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces. 相似文献
19.
Two-stage folding of HP-35 from ab initio simulations 总被引:1,自引:0,他引:1
20.
Cronan JE 《The Journal of biological chemistry》2002,277(25):22520-22527
Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel beta-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits. 相似文献