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1.
Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation 总被引：19，自引：0，他引：19
Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups. 相似文献
2.
Yukie Maruyama Nobuyuki Maruyama Bunzo Mikami Shigeru Utsumi 《Acta Crystallographica. Section D, Structural Biology》2004,60(2):289-297
The crystal structure of the core region of the α′ subunit (α) of soybean β‐conglycinin has been determined at 2.3 Å resolution. α was superimposed on the known crystal structure of the β‐conglycinin β subunit with a small root‐mean square deviation of 0.77 Å, which is consistent with the high sequence identity of 75.5% between α and the β subunit. It is known that the thermal stability of the β subunit is higher than that of the α′ subunit and that their thermal stabilities are conferred by highly homologous core regions. Comparisons of the three‐dimensional structures and primary sequences between α and the β subunit suggest that five factors account for this difference between subunits as regards the difference in thermal stability: (i) the total cavity volume is larger in α, (ii) the cluster of charged residues at the intermonomer interface is smaller in α and α lacks the intermonomer salt bridge of the β subunit, (iii) the solvent‐accessible surface is more hydrophobic in α, (iv) there are fewer proline residues in α and (v) a loop region between helix 3 and strand J′ in α is more flexible owing to the insertion of five additional residues. Although more hydrogen bonds were found in α, this difference should be more than compensated for by the combined contributions of these other factors. 相似文献
3.
Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc. 相似文献
4.
We have recently shown that two of the β‐turns (III and IV) in the ten‐stranded, β‐clam protein, cellular retinoic acid‐binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976–7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side‐chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer‐range stabilizing interactions across the β‐turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a β‐bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long‐range influences in the mechanism of folding of this predominantly β‐sheet protein. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2003 相似文献
5.
6.
The mimicry of protein tertiary folds by chains artificial in backbone chemical composition leads to proteomimetic analogues with potential utility as bioactive agents and as tools, to shed light on biomacromolecule behavior. Notable successes toward such molecules have been achieved; however, as protein structural diversity is vast, design principles must be continually honed as they are applied to new prototype folding patterns. One specific structure where a gap remains in understanding how to effectively generate modified backbone analogues is the metal‐binding β‐turn found in zinc finger domains. The literature precedent suggests several factors that may act in concert, including the artificial moiety used to modify the turn, the sequence in which it is applied, and modifications present elsewhere in the domain. Here, we report efforts to gain insights into these issues and leverage these insights to construct a zinc finger mimetic with backbone modifications throughout its constituent secondary structures. We first conduct a systematic comparison of four turn mimetics in a common host sequence, quantifying relative efficacy for use in a metal‐binding context. We go on to construct a proteomimetic zinc finger domain in which the helix, strands, and turn are simultaneously modified, resulting in a variant with 23% artificial residues, a tertiary fold indistinguishable from the prototype, and a folded stability comparable to the natural backbone on which the variant is based. Collectively, the results reported provide new insights into the effects of backbone modification on the structure and stability of metal‐binding domains and help inform the design of metalloprotein mimetics. 相似文献
7.
Anu M. Mursula Daan M. F. Van Aalten Yorgo Modis J. Kalervo Hiltunen Rik K. Wierenga 《Acta Crystallographica. Section D, Structural Biology》2000,56(8):1020-1023
The purification, crystallization and X‐ray diffraction analysis of Saccharomyces cerevisiaeΔ3‐Δ2‐enoyl‐CoA isomerase is described. Δ3‐Δ2‐Enoyl‐CoA isomerase is a member of the hydratase/isomerase protein family and is an auxiliary enzyme required for the β‐oxidation of unsaturated fatty acids. It is a hexameric enzyme consisting of six identical 32 kDa subunits of 280 residues each. In crystallization trials three crystal forms were obtained, with tetragonal and hexagonal lattices. A 2.5 Å data set was collected from the unliganded hexagonal crystals with an Rmerge of 6.6%. The crystal, with unit‐cell parameters a = 116.0, b = 116.0, c = 122.9 Å, is likely to have P6322 symmetry. 相似文献
8.
9.
Nobuo Okazaki Maki Kumei Miho Manzoku Seiki Kuramitsu Mikako Shirouzu Akeo Shinkai Shigeyuki Yokoyama 《Acta Crystallographica. Section F, Structural Biology Communications》2007,63(3):173-177
TTHA0281 is a hypothetical protein from Thermus thermophilus HB8 that belongs to an uncharacterized protein family, UPF0150, in the Pfam database and to COG1598 in the National Center for Biotechnology Information Database of Clusters of Orthologous Groups. The X‐ray crystal structure of the protein was determined by a multiple‐wavelength anomalous dispersion technique and was refined at 1.9 Å resolution to a final R factor of 18.5%. The TTHA0281 monomer adopts an α‐β‐β‐β‐α fold and forms a homotetramer. Based on the properties and functions of structural homologues of the TTHA0281 monomer, the TTHA0281 protein is speculated to be involved in RNA metabolism, including RNA binding and cleavage. 相似文献
10.
Rajkishor Rai Srinivasarao Raghothama Rajagopalan Sridharan Padmanabhan Balaram 《Peptide Science》2007,88(3):350-361
Designed octapeptides Boc‐Leu‐Val‐Val‐Aib‐DXxx‐Leu‐Val‐Val‐OMe (DXxx = DAla, 3a; DVal, 3c and DPro, 5a ) and Boc‐Leu‐Phe‐Val‐Aib‐DAla‐Leu‐Phe‐Val‐OMe ( 3b ) have been investigated to construct models of a stable type I′ β‐turn nucleated hairpin and to generate systems for investigating helix–hairpin conformational transitions. Peptide 5a , which contains a central Aib‐DPro segment, is shown to adopt a stable type I′ β‐turn nucleated hairpin structure, stabilized by four cross‐strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a β‐hairpin conformation. Replacement of DPro5 by DAla/DVal ( 3a–c ) results in sequences that form β‐hairpins in hydrogen bonding solvents like CD3OH and DMSO‐d6. However, in CDCl3 evidence for population of helical conformations is obtained. Peptide 6b (Boc‐Leu‐Phe‐Val‐Aib‐Aib‐Leu‐Phe‐Val‐OMe), which contains a centrally positioned Aib‐Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl3 and a significant population of both helices and hairpins in CD3OH and DMSO‐d6. The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central β‐turn permits generation of models for robust β‐hairpins and also for the construction of systems that may be used to probe helix–hairpin conformational transitions. © 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 350–361, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
11.
D. Shortle H. S. Chan K. A. Dill 《Protein science : a publication of the Protein Society》1992,1(2):201-215
We develop a model for the reversible denaturation of proteins and for the effects of single-site mutations on the denatured states. The model is based on short chains of sequences of H (hydrophobic) and P (other) monomers configured as self-avoiding walks on the two-dimensional square lattice. The N (native) state is defined as the unique conformation of lowest contact energy, whereas the D (denatured) state is defined as the collection of all other conformations. With this model we are able to determine the exact partition function, and thus the exact native-denatured equilibrium for various solvent conditions, using the computer to exhaustively enumerate every possible configuration. Previous studies confirm that this model shows many aspects of protein-like behavior. The present study attempts to model how the denatured state (1) depends on the amino acid sequence, and (2) is changed by single-site mutations. The model accounts for two puzzling experimental results: (1) the replacement of a polar residue by a hydrophobic amino acid on the surface of a protein can destabilize a native protein, and (2) the "denaturant slope," m = partial delta G/partial c (where c is the concentration of denaturant--urea, guanidine hydrochloride), can sometimes change by as much as 30% due to a single mutation. The principal conclusion of the present study is that, under strong folding conditions, the denatured conformations that are in equilibrium with the native state are not open random configurations. Instead, they are an ensemble of highly compact conformations with a distribution that depends on the residue sequence and that can be substantially altered by single mutations. Most importantly, we conclude that mutations can exert their dominant effects on protein stability by changing the entropy of folding. 相似文献
12.
The bis‐functionalized diamondoid α‐amino acid 2‐aminoadamantane‐2‐carboxylic acid (Adm) has been used as the building block of four Nα‐formyl homo‐dipeptide alkylamide sequences via a solution‐phase Ugi multicomponent reaction approach. The conformers of these peptides have been determined in the crystalline state by X‐ray diffraction to distinguish the influences of the C‐terminal substituent. One of the Adm peptides folds into an open and a hydrogen‐bonded γ‐turn geometry. Moreover, 3D‐structures have been observed featuring two consecutive γ‐turns in an incipient γ‐helical structure, a significantly distorted nonhelical β‐turn, as well as an S‐shaped conformation with opposite helical screw senses. A significant topological variety is thus exhibited by the ‐Adm‐Adm‐ sequences contingent on their C‐terminal substituents, illustrating both the broad conformational potential and the need for further characterization of this sterically bulky residue in explorations of its ϕ, ψ space. 相似文献
13.
Chao Wang Zhongzhou Chen Xia Hong Fangkun Ning Haolin Liu Jianye Zang Xiaoxue Yan Jennifer Kemp Catherine A. Musselman Tatinna G. Kutateladze Rui Zhao Chengyu Jiang Gongyi Zhang 《Acta Crystallographica. Section D, Structural Biology》2014,70(11):2840-2847
Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β‐catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen‐bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular‐dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation. 相似文献
14.
15.
A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space
Kuo-Chen Chou 《Proteins》1995,21(4):319-344
The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc. 相似文献
16.
Appavu Rajagopal Subrayashastry Aravinda Srinivasarao Raghothama Narayanaswamy Shamala Padmanabhan Balaram 《Peptide Science》2011,96(6):744-756
The Aib‐D Ala dipeptide segment has a tendency to form both type‐I′/III′ and type‐I/III β‐turns. The occurrence of prime turns facilitates the formation of β‐hairpin conformations, while type‐I/III turns can nucleate helix formation. The octapeptide Boc‐Leu‐Phe‐Val‐Aib‐D Ala‐Leu‐Phe‐Val‐OMe ( 1 ) has been previously shown to form a β‐hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc‐Phe‐Val‐Aib‐Xxx‐Leu‐Phe‐NHMe ( 2 , 6 ), Boc‐Val‐Aib‐Xxx‐Leu‐NHMe ( 3 , 7 ), and Boc‐Aib‐Xxx‐NHMe ( 4 , 8 ), where Xxx = D Ala, Aib. For peptides with central Aib‐Aib segments, Boc‐Phe‐Val‐Aib‐Aib‐Leu‐Phe‐NHMe ( 6 ), Boc‐Val‐Aib‐Aib‐Leu‐NHMe ( 7 ), and Boc‐Aib‐Aib‐NHMe ( 8 ) helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non‐hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc‐Phe‐Val‐Aib‐D Ala‐Leu‐Phe‐Val‐NHMe ( 2 ) favors helical conformations in CDCl3 and β‐hairpin conformations in CD3OH. The β‐turn conformations (type‐I′/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc‐Aib‐D Ala‐NHMe ( 4 ) and Boc‐Aib‐Aib‐NHMe ( 8 ) in crystals. The tetrapeptide Boc‐Val‐Aib‐Aib‐Leu‐NHMe ( 7 ) adopts an incipient 310‐helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc‐Val‐Aib‐D Ala‐Leu‐NHMe ( 3 ) adopts a novel α‐turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib‐D Ala segment adopts a type‐I′ β‐turn conformation. The observation of an NOE between Val (1) NH↔HNCH3 (5) in CD3OH suggests, that the solid state conformation is maintained in methanol solutions. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 744–756, 2011. 相似文献
17.
《Peptide Science》2017,108(3)
The conformational characteristics of protected homo‐oligomeric Boc‐[β3(R)Val]n‐OMe, n = 1, 2, 3, 4, 6, 9, and 12 have been investigated in organic solvents using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) absorption spectroscopy and circular dichroism (CD) methods. The detailed 1H NMR analysis of Boc‐[β3(R)Val]12‐OMe reveals that the peptide aggregates extensively in CDCl3, but is disaggregated in 20%, (v/v) dimethyl sulfoxide (DMSO) in CDCl3 and in CD3OH. Limited assignment of the N‐terminus NH groups, together with solvent dependence of NH chemical shifts and temperature coefficients provides evidence for 14‐helix conformation in the 12‐residue peptide. FTIR analysis in CHCl3 establishes that the onset of folding and aggregation, as evidenced by NH stretching bands at 3375 cm−1 (intramolecular) and 3285 cm−1 (intermolecular), begins at the level of the tetrapeptide. The observed CD bands, 214 nm (negative) and 198 nm (positive), support 14‐helix formation in the 9 and 12 residue sequences. The folding and aggregation tendencies of homo‐oligomeric α‐, β‐, and γ‐ residues is compared in the model peptides Boc‐[ωVal]n‐NHMe, ω = α, β, and γ and n = 1, 2, and 3. Analysis of the FTIR spectra in CHCl3, establish that the tendency to aggregate at the di and tripeptide level follows the order β > α∼γ, while the tendency to fold follows the order γ > β > α. 相似文献
18.
The two most common methods of measuring the conformational stability of a protein are differential scanning calorimetry and an analysis of solvent denaturation curves by using the linear extrapolation method. In this article, we trace the history of the linear extrapolation method, review how the method is used to measure protein stability, and then discuss some of the other important uses. Proteins 2000;Suppl 4:1–7. © 2000 Wiley‐Liss, Inc. 相似文献
19.
Masahiro Nakajima Ryuta Yoshida Akimasa Miyanaga Hayao Taguchi 《Acta Crystallographica. Section F, Structural Biology Communications》2014,70(10):1398-1401
Lin1840 is a putative β‐glucosidase that is predicted to be involved in 1,2‐β‐glucan metabolism since the lin1839 gene encoding a 1,2‐β‐oligoglucan phosphorylase and the lin1840 gene are located in the same gene cluster. Here, Lin1840 was crystallized. The crystals of Lin1840 diffracted to beyond 1.8 Å resolution. The crystal belonged to space group I121, with unit‐cell parameters a = 89.75, b = 95.10, c = 215.00 Å, α = 90.00, β = 96.34, γ = 90.00°. 相似文献
20.
Bruno Drouillat Cristina Peggion Barbara Biondi Karen Wright Franois Couty Marco Crisma Fernando Formaggio Claudio Toniolo 《Journal of peptide science》2019,25(5)
α‐Amino acid residues with a ?,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an Nα‐alkylated four‐membered ring and Cα‐methylation. We have already reported that (S)‐(αMe)Aze, when followed by (S)‐Ala in the homochiral dipeptide sequential motif ‐(S)‐(αMe)Aze‐(S)‐Ala‐, tends to generate the unprecedented γ‐bend ribbon conformation, as formation of a regular, fully intramolecularly H‐bonded γ‐helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)‐Ala/(R)‐(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type‐II β‐turns or in γ‐turns depending on the experimental conditions. 相似文献