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991.
Gonzalez-Malerva L Cruz-Rivera M Reynoso-Ducoing O Retamal C Flisser A Ambrosio JR 《Cell biology international》2004,28(12):885-894
Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage. 相似文献
992.
Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria
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Casadio R Fariselli P Finocchiaro G Martelli PL 《Protein science : a publication of the Protein Society》2003,12(6):1158-1168
We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species. 相似文献
993.
Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange
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Cristian L Lear JD DeGrado WF 《Protein science : a publication of the Protein Society》2003,12(8):1732-1740
Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins. 相似文献
994.
Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents. 相似文献
995.
Burton RE Baker TA Sauer RT 《Protein science : a publication of the Protein Society》2003,12(5):893-902
ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis. 相似文献
996.
Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system
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Berthold DA Stenmark P Nordlund P 《Protein science : a publication of the Protein Society》2003,12(1):124-134
The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase. 相似文献
997.
Those amazing dinoflagellate chromosomes 总被引:2,自引:0,他引:2
Rizzo PJ 《Cell research》2003,13(4):215-217
Dinoflagellates are a very large and diverse group of eukaryotic algae that play a major role in aquaticfood webs of both fresh water and marine habitats. Moreover, the toxic members of this group posea health threat in the form of red tides. Finally, dinoflagellates are of great evolutionary importance,because of their taxonomic position, and their unusual chromosome structure and composition. While the cytoplasm of dinoflagellates is typically eukaryotic, the nucleus is unique when compared to the nucleusof other eukaryotes. More specifically, while the chromosomes of all other eukaryotes contain histones,dinoflagellate chromosomes lack histones completely. There are no known exceptions to this observation: all dinoflagellates lack histones, and all other eukaryotes contain histones. Nevertheless, dinoflagellates remain a relatively unstudied group of eukaryotes. 相似文献
998.
Membrane glycine transport proteins 总被引:1,自引:1,他引:0
Tunnicliff G 《Journal of biomedical science》2003,10(1):30-36
Structurally, the simplest amino acid is glycine, and it has a number of important yet distinct functions in the body. This review focuses on the different transport systems and the associated carrier proteins for glycine that are responsible for its movement across biological membranes. Transport proteins in the class GLYT appear to be the most specific for glycine. However, the B0+ system also carries significant amounts of glycine. Other amino acid transport systems capable of carrying small amounts of glycine are ASC, asc and system L. In addition, an ATP-dependent transport process exists that takes up glycine into synaptic vesicles at nerve endings. This is known as the vesicular inhibitory amino acid transporter since, in addition to glycine, it can transport possibly two other inhibitory neurotransmitters. 相似文献
999.
Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability. 相似文献
1000.