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1.
    
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   

2.
    
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   

3.
    
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   

4.
    
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   

5.
    
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   

6.
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   

7.
    
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   

8.
    
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   

9.
    
Plasmid‐encoded class C β‐lactamases, including CMY‐1 and CMY‐­10, hydrolyze the lactam bonds of β‐lactam antibiotics, inducing therapeutic failure and a lack of eradication of clinical isolates by third‐generation cephalosporins or cephamycins. Therefore, the enzymes are potential targets for developing agents against pathogens isolated from patients suffering from wound infection, urinary tract infection or pneumonia. CMY‐1 and CMY‐10 were purified and crystallized at 298 K. X‐ray diffraction data from CMY‐1 and CMY‐­10 crystals have been collected to 2.5 and 1.5 Å resolution, respectively, using synchrotron radiation. The crystals of the two proteins are isomorphous and belong to the primitive monoclinic space group P21.  相似文献   

10.
    
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   

11.
    
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12.
    
The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

13.
    
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14.
    
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   

15.
    
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16.
    
Nonprotein amino acids are being extensively used in the design of synthetic peptides to create new structure mimics. In this study we report the effect of methylene group insertions in a heptapeptide Boc‐Ala1‐Leu2‐Aib3‐Xxx4‐Ala5‐Leu6‐Aib7‐OMe which nicely folds into a mixed 310‐/α‐helical structure when Xxx= Ala. Analogs of this peptide have been made and studied by replacing central Xxx4 residue with Glycine (α‐residue), β‐Alanine (β‐Αla), γ‐aminobutyric acid (Gaba), and ε‐aminocaproic acid (ε‐Aca). NMR and circular dichroism were used to study the solution structure of these peptides. Crystals of the peptides containing alanine, β‐Αla, and Gaba reveal that increasing the number of central methylene (‐CH2‐) groups introduces local perturbations even as the helical structure is retained. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 720–732, 2015.  相似文献   

17.
    
β‐d ‐Xylosidases (EC 3.2.1.37) are hemicellulases that hydrolyze short xylooligosaccharides into single xylose units. In this study, the crystallization and preliminary X‐ray analysis of the β‐d ‐xylosidase (XynB1) from Geobacillus stearothermophilus T‐6, a family 39 glycoside hydrolase, are described. XynB1 is a tetrameric protein consisting of four identical subunits of 503 amino acids and with a calculated molecular weight of 58 001 Da. Both the native and the selenomethionine‐containing XynB1 were crystallized by the hanging‐drop vapour‐diffusion method and the crystals were found to belong to space group P212121, with unit‐cell parameters a = 92.7, b = 165.7, c = 311.0 Å. The native crystals diffracted X‐rays to a resolution of 2.1 Å.  相似文献   

18.
    
β‐Phosphoglucomutase (β‐PGM), a 28 kDa monomer, catalyzes the reversible conversion of β‐d ‐glucose‐1‐phosphate to β‐d ‐glucose‐6‐­phosphate in maltose metabolism in a variety of organisms. Sequence analysis of β‐PGM indicates that it is a member of the haloacid dehalogenase (HAD) enzyme superfamily, which evolved to cleave C—Cl, C—P and C—OP bonds in a variety of substrates. β‐­PGM has been crystallized using the hanging‐drop method. Diffraction‐quality crystals of the native protein have been obtained from two conditions, both belonging to space group P212121, with unit‐cell parameters a = 53.67, b = 92.78, c = 111.60 and a = 53.21, b = 57.01, c = 76.11 Å. To solve the phase problem, selenomethionine (SeMet) containing β‐PGM crystals have been grown. The SeMet‐containing crystals diffract to high resolution only when grown by microseeding with native crystals. A three‐wavelength data set has been collected to 2.3 Å on crystals of the SeMet‐substituted β‐PGM. The structure solution is currently being attempted by the multiwavelength anomalous diffraction (MAD) phasing method.  相似文献   

19.
    
β‐Mannosidase from Trichoderma reesei, a 105 kDa glycoprotein, has been crystallized. The crystals belong to the space group P41212 or P43212, with unit‐cell dimensions a = b = 165.86, c = 122.46 Å, and diffract beyond 2.75 Å resolution. X‐ray diffraction data were collected from a frozen crystal on a synchrotron X‐ray source.  相似文献   

20.
    
The structure of the β‐lactamase SME‐1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 Å resolution. The overall structure of SME‐1 is similar to that of other class A β‐lactamases. In the active‐site cavity, most of the residues found in SME‐1 are conserved among class A β‐­lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME‐1 was confirmed by site‐directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other β‐­lactam antibiotics tested. Another striking structural feature found in SME‐1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 Å shorter in SME‐1 compared with other class A β‐­lactamases. Consequently, the SME‐1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A β‐lactamases described so far, suggesting that a significant conformational change may be necessary in SME‐1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl‐enzyme intermediate.  相似文献   

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