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881.
882.
Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants. 总被引:3,自引:1,他引:2
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C. A. Royer C. J. Mann C. R. Matthews 《Protein science : a publication of the Protein Society》1993,2(11):1844-1852
Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
883.
Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels. 总被引:1,自引:1,他引:0
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A. Grove J. M. Tomich T. Iwamoto M. Montal 《Protein science : a publication of the Protein Society》1993,2(11):1918-1930
To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures. 相似文献
884.
Comparison of conformational characteristics in structurally similar protein pairs. 总被引:17,自引:7,他引:10
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T. P. Flores C. A. Orengo D. S. Moss J. M. Thornton 《Protein science : a publication of the Protein Society》1993,2(11):1811-1826
Although it is known that three-dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main-chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of C alpha coordinates but also accessibility, Ooi numbers, secondary structure, and side-chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically. 相似文献
885.
Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. 总被引:8,自引:1,他引:7
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J. Safar P. P. Roller D. C. Gajdusek C. J. Gibbs Jr 《Protein science : a publication of the Protein Society》1993,2(12):2206-2216
The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie. 相似文献
886.
In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV. 总被引:2,自引:1,他引:1
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S. M. Potter W. J. Henzel D. W. Aswad 《Protein science : a publication of the Protein Society》1993,2(10):1648-1663
We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility. 相似文献
887.
Representing an ensemble of NMR-derived protein structures by a single structure. 总被引:1,自引:0,他引:1
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M. J. Sutcliffe 《Protein science : a publication of the Protein Society》1993,2(6):936-944
The usefulness of representing an ensemble of NMR-derived protein structures by a single structure has been investigated. Two stereochemical properties have been used to assess how a single structure relates to the ensemble from which it was derived, namely the distribution of phi psi torsion angles and the distribution of chi 1 torsion angles. The results show that the minimized average structure derived from the ensemble (a total of 11 ensembles from the Brookhaven Protein Data Bank were analyzed) does not always correspond well with this ensemble, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. An alternative method that selects the member of the ensemble which is closest to the "average" of the ensemble has been investigated (a total of 23 ensembles from the Brookhaven Protein Data Bank were analyzed). Although this method selected a structure that on the whole corresponded more closely to the ensemble than did the minimized average structure, this is still not a totally reliable means of selecting a single structure to represent the ensemble. This suggests that it is advisable to study the ensemble as a whole. A study has also been made of the practice of selecting the "best" rather than the most representative member of the ensemble. This too suggests that the ensemble should be studied as a whole. A study of the conformational space occupied by the ensemble also suggests the need to consider the ensemble as a whole, particularly for those ensembles generated with a smaller number of experimentally derived restraints per residue. 相似文献
888.
Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. 总被引:7,自引:6,他引:1
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The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
889.
An automated method for modeling proteins on known templates using distance geometry. 总被引:6,自引:5,他引:1
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S. Srinivasan C. J. March S. Sudarsanam 《Protein science : a publication of the Protein Society》1993,2(2):277-289
We present an automated method incorporated into a software package, FOLDER, to fold a protein sequence on a given three-dimensional (3D) template. Starting with the sequence alignment of a family of homologous proteins, tertiary structures are modeled using the known 3D structure of one member of the family as a template. Homologous interatomic distances from the template are used as constraints. For nonhomologous regions in the model protein, the lower and the upper bounds for the interatomic distances are imposed by steric constraints and the globular dimensions of the template, respectively. Distance geometry is used to embed an ensemble of structures consistent with these distance bounds. Structures are selected from this ensemble based on minimal distance error criteria, after a penalty function optimization step. These structures are then refined using energy optimization methods. The method is tested by simulating the alpha-chain of horse hemoglobin using the alpha-chain of human hemoglobin as the template and by comparing the generated models with the crystal structure of the alpha-chain of horse hemoglobin. We also test the packing efficiency of this method by reconstructing the atomic positions of the interior side chains beyond C beta atoms of a protein domain from a known 3D structure. In both test cases, models retain the template constraints and any additionally imposed constraints while the packing of the interior residues is optimized with no short contacts or bond deformations. To demonstrate the use of this method in simulating structures of proteins with nonhomologous disulfides, we construct a model of murine interleukin (IL)-4 using the NMR structure of human IL-4 as the template. The resulting geometry of the nonhomologous disulfide in the model structure for murine IL-4 is consistent with standard disulfide geometry. 相似文献
890.
X. Weng H. Luecke I. S. Song D. S. Kang S. H. Kim R. Huber 《Protein science : a publication of the Protein Society》1993,2(3):448-458
cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I). 相似文献