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1.
Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.
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R A Steinberg 《Molecular and cellular biology》1984,4(6):1086-1095
A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding. 相似文献
2.
Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains 总被引:2,自引:0,他引:2
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R A Steinberg 《The Journal of cell biology》1983,97(4):1072-1080
A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources. 相似文献
3.
Robert A. Steinberg 《Archives of biochemistry and biophysics》1984,234(2):546-551
The major cAMP-binding proteins isolated from [35S]methionine-labeled S49 mouse lymphoma cells or MDBK bovine kidney cells correspond in isoelectric point and apparent molecular weight to the regulatory subunit (R) of type I cAMP-dependent protein kinase. These proteins were compared directly by two-dimensional gel electrophoresis and by two-dimensional gel electrophoresis of peptides generated either from native R with thermolysin and chymotrypsin or from denatured R with papain. Both the undigested proteins and all their major peptides were identical in charge and apparent molecular weights, indicating a very high degree of structural homology. 相似文献
4.
A domain of synapsin I involved with actin bundling shares immunologic cross-reactivity with villin 总被引:3,自引:0,他引:3
Synapsin I is a neuronal phosphoprotein that can bundle actin filaments in vitro. This activity is under phosphorylation control, and may be related to its putative in vivo role of regulating the clustering and release of small synaptic vesicles. We have compared human and bovine synapsin I by peptide mapping, and have used NTCB (2-nitro-5-thiocyano benzoic acid) cleavage to generate a series of peptide fragments from bovine synapsin I. After chymotryptic digestion, 88% of the tyrosine-containing fragments appear to be structurally identical in human and bovine synapsin I, as judged by their positions on high-resolution two-dimensional peptide maps. The alignment of the NTCB peptides within the parent protein have been determined by peptide mapping, and the ability of these fragments to precipitate with actin bundles has been measured. Only peptides that are derived from regions near the ends of the protein are active. One such 25-kDa peptide which sediments with actin also cross-reacts with antibodies to chicken villin, an actin binding and bundling protein derived from the intestinal microvillus. Since in other respects villin appears to be an unrelated protein, these results suggest the possibility that certain actin binding proteins may show immunologic cross-reactivity due to convergent evolution within the acting binding domain. 相似文献
5.
The structural basis of ankyrin function. I. Identification of two structural domains 总被引:7,自引:0,他引:7
The structure of ankyrin, a major linking protein between spectrin and the erythrocyte membrane, was analyzed after restricted proteolytic digestion at 0 degree C. By the use of two-dimensional peptide mapping, we found that tryptic digestion of ankyrin (1 h, 0 degree C) resulted in the production of two nonoverlapping peptides of molecular weights 82,000 and 55,000. The 82,000-dalton peptide had a basic isoelectric point (7.9) and was remarkably sensitive to further proteolytic digestion; after 24 h at 0 degree C, trypsin completely digested this peptide into fragments too small to detect by gel electrophoresis. The 55,000-dalton peptide was neutral (isoelectric point = 6.9-7.2) and more resistant to further proteolytic cleavage. After a 24-h digestion with trypsin at 0 degrees C, the 55,000-dalton peptide was cleaved into two complementary fragments of molecular weight 32,000 and 15,000. Analysis of phosphorylated ankyrin indicated that the phosphates were exclusively found in these two complementary peptides. By comparison with larger fragments, we were able to align the constituent peptides of ankyrin and propose a low resolution model. Ankyrin appears to be a bipolar molecule containing a basic domain of 82,000 daltons and a neutral phosphorylated domain of 55,000 daltons. 相似文献
6.
A Comparative Structural Analysis of the Flagellin Monomers of Caulobacter crescentus Indicates that These Proteins Are Encoded by Two Genes 总被引:13,自引:6,他引:7
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The flagellum of Caulobacter crescentus is composed of two flagellin polypeptide monomers which are distinguished by molecular weight and are closely related by biochemical and immunological criteria (C. Lagenaur and N. Agabian, J. Bacteriol. 132:731-733, 1977). The synthesis and assembly of these two flagellin proteins are developmentally regulated, and the periodicity of expression for each is distinct (C. Lagenaur and N. Agabian, J. Bacteriol. 135:1062-1069, 1978; M. A. Osley, M. Sheffery, and A. Newton, Cell 12:393-400, 1977). To understand the genetic and functional relationship between the 25,000- and 27,500-molecular-weight flagellins of C. crescentus CB15, a detailed comparative analysis of their protein structure was made, using a number of techniques, including one- and two-dimensional peptide mapping, a novel procedure of peptide alignment, and amino terminal amino acid sequence analysis. The tryptic peptides generated by each of the flagellins were compared by two-dimensional thin-layer chromatography. This peptide map analysis indicated that approximately 36% of the peptides generated from these two proteins had similar migration properties. Together with biochemical and immunological criteria, the two-dimensional peptide map suggested some structural relatedness between the monomers. However, a comparison of peptide fragments generated during partial protease digestion of each protein by a method of one-dimensional mapping indicated that the two proteins are structurally unique. A peptide alignment technique was developed to directly compare the primary structure of these proteins. In the peptide alignment procedure the amino terminus of each protein is radioactively labeled. After partial enzymatic digestion, the peptides are fractionated by polyacrylamide gel electrophoresis: those labeled at the amino terminus are then resolved by subsequent autoradiography. Each digest contains a family of amino-terminal-labeled fragments, the sizes of which reflect the sequential alignment of cleavage sites in the protein. A comparison of the alignment of specific cleavage sites of the two flagellins by this technique further established that each flagellin is structurally unique, particularly in the carboxyl terminal region. Finally, comparison of the amino terminal amino acid sequences indicated that the amino terminal region of both flagellins is highly conserved, but that the two polypeptides are clearly not identical. These findings strongly indicate that the two flagellins are encoded by distinct genetic loci and are not the product of novel processing of a single larger precursor. 相似文献
7.
It is an established fact that allelic variation and post-translational modifications create different variants of proteins, which are observed as isoelectric and size subspecies in two-dimensional gel based proteomics. Here we explore the stromal proteome of spinach and Arabidopsis chloroplast and show that clustering of mass spectra is a useful tool for investigating such variants and detecting modified peptides with amino acid substitutions or post-translational modifications. This study employs data mining by hierarchical clustering of MALDI-MS spectra, using the web version of the SPECLUST program (http://bioinfo.thep.lu.se/speclust.html). The tool can also be used to remove peaks of contaminating proteins and to improve protein identification, especially for species without a fully sequenced genome. Mutually exclusive peptide peaks within a cluster provide a good starting point for MS/MS investigation of modified peptides, here exemplified by the identification of an A to E substitution that accounts for the isoelectric heterogeneity in protein isoforms. 相似文献
8.
Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform 总被引:2,自引:1,他引:1
A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit. 相似文献
9.
Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis 总被引:3,自引:0,他引:3
Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs. 相似文献
10.
W W Barrington K A Jacobson G L Stiles 《The Journal of biological chemistry》1989,264(22):13157-13164
A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease. These data suggest that agonist and antagonist photoaffinity probes interact with an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation. 相似文献
11.
The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility. 相似文献
12.
Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs. 相似文献
13.
Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli. 总被引:5,自引:3,他引:2
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Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes. 相似文献
14.
B P Sokolov B M Sher V N Kalinin 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1987,(3):43-45
Two highly efficient methods of CNBr-peptide mapping of polypeptides divided by polyacrylamide gel electrophoresis are described. The first is elaborated on the basis of peptide mapping of collagen proposed by G. Barsh et al. The following three modifications diminish wasting the material essential for the method. 1. CNBr treatment takes place in the absence of CNBr solution outside the gel, excluding the peptides elution from the gel fragments in the process of mapping. 2. After CNBr treatment the solution of CNBr is substituted by the samples buffer before electrophoresis by means of drying and subsequent addition of minimal volumes of the buffer. The latter procedures substitute the gel washing out by the buffer solution. 3. The step of washing the gel fragments by the 70% strong solution of formic acid before CNBr treatment is excluded. The second method of CNBr-peptide mapping is notable for extracting peptides from the gel fragments in the process of CNBr-treatment and permits obtaining of the high quality peptide electrophoregrams. 相似文献
15.
Partial digestion of the native beta subunit of F1-ATPase from the thermophilic Bacillus strain PS3 by three different proteases produced a limited number of peptide fragments. In most cases, the peptides remained associated, and the gross structure of the beta subunit was not destroyed. Furthermore, most peptides were able to reassociate into the form of the beta subunit after denaturating urea treatment. Therefore, the cleaved sites are most likely located in water-exposed loop regions in the tertiary structure of the protein. Almost all peptides were analyzed, and 17 cleaved sites were determined. From the analysis of the distribution of cleaved sites and deletions or insertions in the multiple amino acid sequence alignment of proteins homologous to the beta subunit, locations of five loops and four candidate loops in the beta subunit are suggested. There are two large loops in the central region of the beta subunit sequence, and dicyclohexylcarbodiimide-reactive Glu190 is located in one of them. Tyr341, involved in putative catalytic ATP binding, is also found in one of the loops. Then, taking cleaved sites as a reference, two kinds of expression plasmids, each of which carried genes of two complementary peptide fragments, 1-193 and 198-473 or 1-284 and 285-473, were constructed and expressed in Escherichia coli. For each plasmid, two peptides were coexpressed, associated into a stable beta subunit form in E. coli cells, and purified without dissociation. When these beta subunits were denatured by urea and applied to polyacrylamide gel without denaturant, a protein band with the same mobility as that of the beta subunit appeared, indicating that reassociation of peptide fragments into the form of the beta subunit occurred upon removal of urea. These beta subunits retained the ability to reconstitute the alpha 3 beta 3 gamma complexes even though the efficiency of reconstitution and the recovered ATPase activities were decreased. These complexes were stable at high or low temperature, and ATPase activities were sensitive to inhibition by N3-. 相似文献
16.
Stefan Bühler Jenny Michels Silke Wendt Alexander Rück Dieter Brdiczka Wolfram Welte Michael Przybylski 《Proteins》1998,33(Z2):63-73
Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63–73, 1998. © 1998 Wiley-Liss, Inc. 相似文献
17.
Peptide mapping of heterogeneous protein samples. 总被引:5,自引:0,他引:5
A simple two-dimensional electrophoretic method for peptide mapping of heterogeneous protein samples is presented. The reduced and denatured proteins of the mixture are separated in a first dimension by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After completion of the electrophoresis, the whole gel lane is equilibrated in stacking gel buffer and is transferred at right angles onto a second slab gel. A protease solution is overlayed on the gel lane and a partial proteolysis of the proteins to be analyzed is performed during the stacking phase of the second electrophoresis. The second electrophoresis resolves the characteristic pattern of peptides of each individual protein as a series of spots located below the original position of the undigested protein. The peptide maps of the following samples are presented as examples: protein P23 and P23* of bacteriophage T4, membranes of Dictyostelium discoideum, membranes of human erythrocytes, and 35S-labeled proteins of D. discoideum synthesized in vivo or in a cell-free wheat germ extract. In complex samples, up to 20 individual proteins can be analyzed at once and a protein comprising only 1% of the total sample generates a clearly identifiable peptide pattern. Good reproducibility of the patterns obtained allows the comparison of samples of different origins. 相似文献
18.
Paola Strocchi Victor S. Sapirstein † Charles S. Rubin ‡ Jeffrey M. Gilbert 《Journal of neurochemistry》1984,43(2):466-471
Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Ⅰ型膜融合蛋白(class I membrane fusion protein)在Ⅰ型包膜病毒入侵宿主细胞过程中发挥重要作用。基于抑制六螺旋结构形成的多肽类融合抑制剂设计的原理是模拟该蛋白融合区域的自身序列,与病毒融合蛋白结合形成异源六聚体,从而阻断病毒与靶细胞膜的融合。此类融合抑制剂的传统设计主要基于一级与二级结构,但为进一步强化其抗病毒活性,通常需依赖病毒膜融合蛋白三级结构信息,从而限制了对那些尚无病毒蛋白三级结构信息的新发病毒的多肽类融合抑制剂的快速优化和研发。本研究提出了不依赖蛋白三级结构信息,而利用I-Mutant2.0软件来辅助设计和优化多肽类病毒膜融合抑制剂的设想。根据I-Mutant2.0的预测结果,以中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)为模型,分析该病毒HR2区融合抑制剂序列中若干适合与不适合优化的位点,并设计了一系列多肽。结果发现,对适合优化的位点进行调整的多肽,其对HR1的结合能力及对病毒的抑制活性均有所提升;反之,多肽活性明显下降。结果表明,利用I-Mutant2.0辅助设计与优化病毒融合抑制多肽的方法具有一定的可行性,为进一步开发新的融合抑制剂设计方法奠定了基础。 相似文献
20.
Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique 总被引:21,自引:0,他引:21
R B Pepinsky 《The Journal of biological chemistry》1983,258(18):11229-11235
In HTG2 hamster cells infected with the replication-defective Gazdar murine sarcoma virus only immature virus particles are formed, with the uncleaved gag precursor Pr65 as the only major protein in the virion. We have investigated the structure of these particles by using in situ cross-linking followed by chemical and enzymatic cleavages of Pr65 to localize sites of lipid-protein and protein-protein interactions. Lipid-protein cross-links were localized within a 10-kDa fragment in the p15 region of Pr65. Homotypic protein-protein cross-links between Pr65 units were localized within the p15 regions and also within the p10 regions of Pr65. Similar data for processed gag proteins in Rauscher murine leukemia virus, a prototype of a mature C-type virus, suggest that these interactions of the gag precursor are not altered during maturation. To identify the sites of cross-linking within Pr65, we have developed a two-dimensional peptide mapping technique that is based on nearest neighbor analysis of fragments released by cyanogen bromide treatment of partial cleavage products in gel slices. In conjunction with cross-linking, the peptide mapping technique is a powerful means for localizing specific interactions on a polypeptide backbone. 相似文献