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1.
Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial
structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight
oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax
during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation,
chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions
of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed
by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar
upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed. 相似文献
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Recently, we have developed a method (Shih et al., Proteins: Structure, Function, and Bioinformatics 2007;68: 34-38) to compute correlation of fluctuations of proteins. This method, referred to as the protein fixed-point (PFP) model, is based on the positional vectors of atoms issuing from the fixed point, which is the point of the least fluctuations in proteins. One corollary from this model is that atoms lying on the same shell centered at the fixed point will have the same thermal fluctuations. In practice, this model provides a convenient way to compute the average dynamical properties of proteins directly from the geometrical shapes of proteins without the need of any mechanical models, and hence no trajectory integration or sophisticated matrix operations are needed. As a result, it is more efficient than molecular dynamics simulation or normal mode analysis. Though in the previous study the PFP model has been successfully applied to a number of proteins of various folds, it is not clear to what extent this model will be applied. In this article, we have carried out the comprehensive analysis of the PFP model for a dataset comprising 972 high-resolution X-ray structures with pairwise sequence identity or=0.5. Our result shows that the fixed-point model is indeed quite general and will be a useful tool for high throughput analysis of dynamical properties of proteins. 相似文献
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Lardinois OM Medzihradszky KF Ortiz de Montellano PR 《The Journal of biological chemistry》1999,274(50):35441-35448
Lactoperoxidase (LPO) reacts with H(2)O(2) to sequentially give two Compound I intermediates: the first with a ferryl (Fe(IV)=O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitroso-benzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H(2)O(2)-dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H(2)O(2) is fully active but no longer forms dimers when incubated with H(2)O(2), clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H(2)O(2)-dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates. 相似文献
4.
Activities of the Sex-lethal protein in RNA binding and protein:protein interactions. 总被引:7,自引:0,他引:7
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The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain. 相似文献
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Goldstein RA 《Current opinion in structural biology》2008,18(2):170-177
The observed distribution of protein structures can give us important clues about the underlying evolutionary process, imposing important constraints on possible models. The availability of results from an increasing number of genome projects has made the development of these models an active area of research. Models explaining the observed distribution of structures have focused on the inherent functional capabilities and structural properties of different folds and on the evolutionary dynamics. Increasingly, these elements are being combined. 相似文献
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Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrPC) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed. 相似文献
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The mechanosensitive channel of large conductance (MscL) belongs to a family of transmembrane channel proteins in bacteria and functions as a safety valve that relieves the turgor pressure produced by osmotic downshock. MscL gating can be triggered solely by stretching of the membrane. This work reports an effort to understand this mechanotransduction by means of molecular dynamics (MD) simulation on the MscL of mycobacterium tuberculosis embedded in a palmitoyloleoylphosphatidylethanolamine membrane. Equilibrium MD under zero membrane tension produced a more compact protein structure, as measured by its radii of gyration, compared to the crystal structure, in agreement with previous experimental findings. Even under a large applied tension up to 1000 dyn/cm, the MscL lateral dimension largely remained unchanged after up to 20 ns of simulation. A nonequilibrium MD simulation of 3% membrane expansion showed a significant increase in membrane rigidity upon MscL inclusion, which can contribute to efficient mechanotransduction. Direct observation of channel opening was possible only when an explicit lateral bias force was applied to each of the five subunits of MscL in the radially outward direction. Using this force, open structures with a large pore of radius 10 Å could be obtained. The channel opening takes place in a stepwise manner and concurrently with the water chain formation across the channel, which occurs without direct involvement of protein hydrophilic residues. The N-terminal S1 helices stabilize the open structure, and the membrane asymmetry (different lipid density on the two leaflets of membrane) promotes channel opening. 相似文献
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Garcia-Saez I Lacroix FB Blot D Gabel F Skoufias DA 《Journal of molecular biology》2011,405(2):331-340
Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here we report the crystal structure of the shortest isoform of HBXIP (91 aa long, ∼ 11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a superfamily of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended β-sheet area formed by 10 anti-parallel β-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is predominantly in dimeric form in solution. The fit between the experimental small-angle X-ray scattering curve and the backcalculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP. 相似文献
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Wilder PT Lin J Bair CL Charpentier TH Yang D Liriano M Varney KM Lee A Oppenheim AB Adhya S Carrier F Weber DJ 《Biochimica et biophysica acta》2006,1763(11):1284-1297
S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex. 相似文献
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Sigoillot FD Kotsis DH Serre V Sigoillot SM Evans DR Guy HI 《The Journal of biological chemistry》2005,280(27):25611-25620
CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus. 相似文献
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Brown JA Eberhardt DM Schrick FN Roberts MP Godkin JD 《Molecular reproduction and development》2003,64(3):261-269
Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function. 相似文献
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Characterization of the arabidopsis formin-like protein AFH1 and its interacting protein 总被引:8,自引:0,他引:8
A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA. The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins. In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmembrane domain suggesting its association with membrane. Cell fractionation by differential centrifugation demonstrated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction. An Arabidopsis cDNA library was screened to identify proteins that interact with the C-terminal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2. Experiments using recombinant proteins expressed in E. coli demonstrated that FIP2 interacted directly with AFH1. The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases. AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton. 相似文献
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Simpson F Martin S Evans TM Kerr M James DE Parton RG Teasdale RD Wicking C 《Traffic (Copenhagen, Denmark)》2005,6(6):442-458
The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains. 相似文献
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