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1.
    
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   

2.
    
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   

3.
    
While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔGU = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. 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   

4.
    
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   

5.
    
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   

6.
    
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   

7.
    
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   

8.
    
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   

9.
    
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   

10.
    
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   

11.
    
Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.  相似文献   

12.
    
The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I possesses at least 35 putative genes encoding enzymes that belong to the α/β‐hydrolase superfamily. One of those genes, the metallo‐hydrolase‐encoding igni18, was cloned and heterologously expressed in Pichia pastoris. The enzyme produced was purified in its catalytically active form. The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of 2.3 Å. The crystal belonged to space group R32, with unit‐cell parameters a = b = 67.42, c = 253.77 Å, α = β = 90.0, γ = 120.0°. It is suggested that it contains one monomer of Igni18 within the asymmetric unit.  相似文献   

13.
    
An important nucleation event during the folding of staphylococcal nuclease involves the formation of a β‐hairpin by the sequence 21DTVKLMYKGQPMTFR35. Earlier studies show that the turn sequence ‘YKGQP’ has an important role in the folding of this β‐hairpin. To understand the active or passive nature of the turn sequence ‘YKGQP’ in the folding of the aforementioned β‐hairpin sequence, we studied glycine mutant peptides Ac‐2DTVKLMYGGQPMTFR16‐NMe (K9G:15), Ac‐2DTVKLMYKGGPMTFR16‐NMe (Q11G:15), Ac‐2DTVKLMYGGGPMTFR16‐NMe (K9G/Q11G:15), and Ac‐2DTVKLMGGGGGMTFR16‐NMe (penta‐G:15) by using molecular dynamics simulations, starting with two different unfolded states, polyproline II and extended conformational forms. Further, 5mer mutant turn peptides Ac‐2YGGQP6‐NMe (K3G:5), Ac‐2YKGGP6‐NMe (Q5G:5), Ac‐2YGGGP6‐NMe (K3G/Q5G:5), and Ac‐2GGGGG6‐NMe (penta‐G:5) were also studied individually. Our results show that an initial hydrophobic collapse and loop closure occurs in all 15mer mutants, but only K9G:15 mutant forms a stable native‐like β‐hairpin. In the other 15mer mutants, the hydrophobic collapsed state would not proceed to β‐hairpin formation. Of the different simulations performed for the penta‐G:15 mutant, in only one simulation a nonnative β‐hairpin conformation is sampled with highly flexible loop region (8GGGGG12), which has no specific conformational preference as a 5mer. While the sequence ‘YGGQP’ in the K3G:5 simulation shows relatively higher β‐turn propensity, the presence of this sequence in K9G:15 peptide seems to be driving the β‐hairpin formation. Thus, these results seem to suggest that for the formation of a stable β‐hairpin, the initial hydrophobic collapse is to be assisted by a turn propensity. Initial hydrophobic collapse alone is not sufficient to guide β‐hairpin formation. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

14.
    
The X‐ray crystal structure of AmpC β‐lactamase (AmpCD) with a tripeptide deletion (Gly286‐Ser287‐Asp288) produced by Escherichia coli HKY28, a ceftazidime‐resistant strain, was determined at a resolution of 1.7 Å. The structure of AmpCD suggests that the tripeptide deletion at positions 286–288 located in the H10 helix causes a structural change of the Asn289–Asn294 region from the α‐helix present in the native AmpC β‐lactamase of E. coli to a loop structure, which results in a widening of the substrate‐binding site.  相似文献   

15.
The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.  相似文献   

16.
    
To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H3C—(CH2)14—CONH—; Pda: —CONH— (CH2)14—CH3) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic 13C NMR chemical‐shifts of the Pro Cβ and Cγ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and 1H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of 1H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.  相似文献   

17.
    
β‐Microseminoprotein (β‐MSP) is a small cysteine‐rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino‐acid sequences of β‐MSP proteins suggests that the protein is a rapidly evolving protein. The function of β‐MSP is poorly understood. Furthermore, no crystal structure has been reported of any β‐MSP; therefore, determination of the crystal structure of β‐MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X‐ray diffraction analysis of β‐MSP from human seminal plasma are described. The protein was purified using anion‐exchange and size‐exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4322 and contained three β‐MSP molecules in the asymmetric unit. X‐ray intensity data were collected to 2.4 Å resolution.  相似文献   

18.
    
The preferred conformations of peptides heavily based on the currently extensively exploited achiral and chiral α‐amino acids with a quaternary α‐carbon atom, as determined by conformational energy computations, crystal‐state (x‐ray diffraction) analyses, and solution (1H‐NMR and spectroscopic) investigations, are reviewed. It is concluded that 310/α‐helical structures and the fully extended (C5) conformation are preferentially adopted by peptide sequences characterized by this family of amino acids, depending upon overall bulkiness and nature (e.g., whether acyclic or C ↔ C cyclized) of their side chains. The intriguing relationship between α‐carbon chirality and bend/helix handedness is also illustrated. γ‐Bends and semiextended conformations are rarely observed. Formation of β‐sheet structures is prevented. © 2002 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 60: 396–419, 2001  相似文献   

19.
    
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   

20.
    
β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β3‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β3h‐D ‐Ala in position 2 or β3hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

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