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1.
Oxidative folding is the concomitant formation of the native disulfide bonds and the native tertiary structure from the reduced and unfolded polypeptide. Of interest is the inherent conformational tendency (bias) present in the reduced polypeptide to dictate the formation of the full set of native disulfide bonds. Here, by application of a novel tool, we have been able to assess this "native conformational tendency" present in reduced and unfolded bovine pancreatic ribonuclease A (RNase A). The essence of this method lies in the ability of the oxidant [Pt(en)(2)Cl(2)](2+) (where "en" is ethylenediamine) to oxidize disulfide bonds under conditions in which both reduction and disulfide reshuffling, which are essential for rearranging non-native disulfide bonds, are extremely slow. When applied to RNase A, the method revealed little or no bias toward formation of the full native set of disulfide bonds in the fully reduced protein. 相似文献
2.
Decorsin,an antagonist of integrin glycoprotein IIb/IIIa,contains Arg-Gly-Asp(RGD)sequence and three disulfide bridges.The function of RGD sequence has already been well defined,but the roles of conserved disulfide bonds in antihemostatic proteins still remain unclear.Herein we use the fusion expression and characterization of mutant decorsin to study the functions of disulfide bonds in protein structure,stability and biological activity.The purified protein shows an apparent inhibition of activity to platelet aggregation induced by ADP with IC50 of 500 nM.The removal of cys7-cys15(from cysteine to serine)at the N-terminal causes a thirty-fold decrease of the inhibition activity with IC50 of 15 M,whereas the mutation of cys22-cys38 at the C-terminal completely impairs the biological activity of decorsin.The overall secondary and tertiary structures of decorsin are disrupted inevitably without disulfide bonds.Using a domain insertion mutation,the retaining of RGD loop and the adjacent disulfide bond produces a week antihemostatic activity of decorsin.This reveals that the overall structure of decorsin stabilized by the three conserved disulfide bridges is cooperative for antihemostatic function.Our study on the effect of disulfide bonds together with RGD-sequence on the protein function is helpful for structure-based drug design of antithrombotic research. 相似文献
3.
Rheological properties of wheat gluten as a function of the presence of free sulfhydryls and disulfide bonds in addition to
their ratio during various conditioning regimes were studied. It was shown that coagulation of the gluten as the temperature
increased was accompanied by a decrease in the number of free sulfhydryls and an increase in the number of rheologically active
disulfide bonds. Changes occurring in the gliadin and glutenin fractions during various conditioning regimes were seen using
IR spectroscopy. It was found that the relative strength of absorption bands due to H-bonds increased as the treatment temperature
was raised above 60°C. Significant changes in the protein molecule structure that caused substantial changes in its rheological
properties because of thiol–disulfide exchange reactions and H-bond strength occurred during the conditioning. 相似文献
4.
Cole SR Ma X Zhang X Xia Y 《Journal of the American Society for Mass Spectrometry》2012,23(2):310-320
The fragmentation chemistry of peptides containing intrachain disulfide bonds was investigated under electron transfer dissociation
(ETD) conditions. Fragments within the cyclic region of the peptide backbone due to intrachain disulfide bond formation were
observed, including: c (odd electron), z (even electron), c-33 Da, z + 33 Da, c + 32 Da, and z–32 Da types of ions. The presence
of these ions indicated cleavages both at the disulfide bond and the N–Cα backbone from a single electron transfer event.
Mechanistic studies supported a mechanism whereby the N–Cα bond was cleaved first, and radical-driven reactions caused cleavage
at either an S–S bond or an S–C bond within cysteinyl residues. Direct ETD at the disulfide linkage was also observed, correlating
with signature loss of 33 Da (SH) from the charge-reduced peptide ions. Initial ETD cleavage at the disulfide bond was found
to be promoted amongst peptides ions of lower charge states, while backbone fragmentation was more abundant for higher charge
states. The capability of inducing both backbone and disulfide bond cleavages from ETD could be particularly useful for sequencing
peptides containing intact intrachain disulfide bonds. ETD of the 13 peptides studied herein all showed substantial sequence
coverage, accounting for 75%–100% of possible backbone fragmentation. 相似文献
5.
Jean-Pierre Duneau Serge Crouzy Yves Chapron Monique Genest 《Theoretical chemistry accounts》1999,101(1-3):87-91
The structure and dynamics of the ErbB-2 transmembrane domain have been examined using molecular dynamics techniques both
in vacuum and within an explicit hydrated L-α-dilauroyl-phosphatidyl-ethanolamine environment. In-vacuum simulations show that a highly cooperative structural transition
occurs frequently within the α-helical transmembrane domain which converts to local π-helices. We show that the α-helix alteration
does not depend upon the force field or initial side-chain conformations but is intimately related to the sequence. The membrane-like
environment does not prevent the structural transition in the helix but slows down the peptide dynamics indicating that the
appearance of a π-bulge is not an artifact of the vacuum approximation. The consequences of π-helix formation could be very
huge for the ErbB-2 receptor which is involved in numerous human cancers and also for other membrane proteins wherein similar
local structures are also observed experimentally.
Received: 9 May 1998 / Accepted: 3 September 1998 / Published online: 17 December 1998 相似文献
6.
Ilario Losito Elisabetta Stringano Saverio Carulli Francesco Palmisano 《Analytical and bioanalytical chemistry》2010,396(6):2293-2306
The Maillard-reaction-induced lactosylation of the major whey proteins, α-lactalbumin (α-La) and β-lactoglobulins (β-Lg) A
and B, occurring upon heating at 70, 80 and 90 °C for 1 to 5 h in the presence of lactose excess, was studied by HPLC coupled
to electrospray ionization single and tandem mass spectrometry (HPLC-ESI-MS, MS/MS). The presence of significant amounts of
mono and bi-lactosylated forms of the three proteins and their increase with heating temperature and time were assessed from
MS data. Evidences for a concomitant, significant denaturation, involving partial tertiary structure unfolding, were also
obtained in the case of β-lactoglobulins. A subsequent ESI-MS and MS/MS investigation on the tryptic digests of heated protein
solutions exhibiting high percentages of mono and bi-lactosylated forms provided information on lactosylation sites. In particular,
the latter were identified both on tryptic and on aspecific peptides, whose unusual relevance (compared to similar studies)
was found to be due mainly to heat-induced protein degradation, occurring before protein digestion with trypsin. Among lactosylation
sites identified only on tryptic peptides, i.e., those reasonably related to intact protein lactosylation, two lysines residues
were found for α-La, both located in accessible regions of its tertiary structure. In the case of β-Lg, besides three sites
common to variants A and B (leucine 1, lysines 70, and 75), lysine 69 was found to be lactosylated only in variant B. Its
proximity to a critical region of β-Lg tertiary structure suggests that the difference between the two variants could be ascribed
to a different evolution of their conformation upon heating. 相似文献
7.
Polymer coiled coils are composed of entangled linear chains in a helical conformation. Their mechanical characteristics are
interesting because these structures are involved in the composition of natural fibrillar structures. The method of molecular
dynamics is used for the simulation of stretching at a constant rate for a superhelical fragment of myosin protein composed
of two identical α helices containing 126 amino acid residues in each helix. The case of shear deformation of a molecule is
considered (the load is applied to the N terminus of one chain and to the C terminus of another chain). In this case of loading,
slippage of chains with respect to each other can occur. Deformation of a molecule proceeds in several stages. At the initial
stage, the superhelix is unfolded and there is a gradual unfolding of end fragments of individual α helices; this process
is accompanied by their displacement with respect to the helical fragment of the neighboring chain. In this case, the reaction
force increases. At the second stage of stretching, the process passes to the mechanism of deformation when, in the central
part of the molecule, α-helical fragments of both chains unfold. In this region, the reaction-force-extension curve shows
a plateau region. Between unfolded fragments, new hydrophobic contacts and hydrogen bonds are formed, and fragments of the
β structure emerge. Once all turns of α helices in the central parts of the molecule are unfolded, the mechanism of deformation
changes and further extension of a molecule proceeds via straightening of previously unfolded central fragments, a process
that is accompanied by an increase in the reaction force. When chains achieve their limiting extension, slippage commences
with an accompanying decrease in the reaction force. 相似文献
8.
Lu Sheng Jiaping Chen Jing Li Weiyun Zhang 《Applied biochemistry and biotechnology》2011,163(5):669-678
The exopolysaccharide (EPS) is a polysaccharide from cultivated Cordyceps sinensis, which possesses immunomodulatory and antitumor effects, was purified by DEAE-32 cellulose and Sephadex G-200 gel. The preliminary
characters of EPS were analyzed by IR and GC, and the molecular weight was estimated by gel filtration. The effect of EPS
on proliferation ability of lymphocytes from ICR mice was assayed by MTT method. The mRNA and protein expression levels of
several cytokines in spleen and thymus cells were detected by RT-PCR and ELISA. The results showed that EPS consists of mannose,
glucose, and galactose in a ratio of 23:1:2.6. Its molecular weight is about 1.04 × 105. EPS elevated proliferation ability of spleen lymphocytes only at 100 μg/ml after 48 h treatment. Tumor necrosis factor alpha
(TNF-α), interferon-α (IFN-γ), and interleukin-2 (IL-2) mRNA levels in splenocytes and thymocytes were increased after EPS
treatment for 2, 4, 8, or 20 h. EPS also significantly elevated splenic TNF-α and IFN-γ protein expressions at 100 μg/ml and
increased thymic TNF-α and IFN-γ protein levels at 50 and 100 μg/ml. These data indicated that EPS may stimulate cytokine
expressions of immunocytes. 相似文献
9.
Silvers R Sziegat F Tachibana H Segawa S Whittaker S Günther UL Gabel F Huang JR Blackledge M Wirmer-Bartoschek J Schwalbe H 《Journal of the American Chemical Society》2012,134(15):6846-6854
During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale. 相似文献
10.
Hongmei Yang Ning Liu Xiaoyan Qiu Shuying Liu 《Journal of the American Society for Mass Spectrometry》2009,20(12):2284-2293
A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts
is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups
within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]+ or [M+n+n′ matrix+Na]+ (n = the number of cysteine residues, n′=1, 2,…, n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic
acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis
of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of
189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid,
ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline
conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several
factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast,
effective, and robust to identify disulfide bonds in proteins/peptides. 相似文献
11.
Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily 下载免费PDF全文
Ho Yeung Dr. Christopher J. Squire Yuliana Yosaatmadja Dr. Santosh Panjikar Gemma López Prof. Dr. Antonio Molina Prof. Dr. Edward N. Baker Dr. Paul W. R. Harris Prof. Dr. Margaret A. Brimble 《Angewandte Chemie (International ed. in English)》2016,55(28):7930-7933
Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. 相似文献
12.
Dr. Olatz Larrañaga Prof. Dr. Ana Arrieta Prof. Dr. Célia Fonseca Guerra Prof. Dr. F. Matthias Bickelhaupt Dr. Abel de Cózar 《化学:亚洲杂志》2021,16(4):315-321
We have quantum chemically studied the structure and nature of alkali- and coinage-metal bonds (M-bonds) versus that of hydrogen bonds between A−M and B− in archetypal [A−M⋅⋅⋅B]− model systems (A, B=F, Cl and M=H, Li, Na, Cu, Ag, Au), using relativistic density functional theory at ZORA-BP86-D3/TZ2P. We find that coinage-metal bonds are stronger than alkali-metal bonds which are stronger than the corresponding hydrogen bonds. Our main purpose is to understand how and why the structure, stability and nature of such bonds are affected if the monovalent central atom H of hydrogen bonds is replaced by an isoelectronic alkali- or coinage-metal atom. To this end, we have analyzed the bonds between A−M and B− using the activation strain model, quantitative Kohn-Sham molecular orbital (MO) theory, energy decomposition analysis (EDA), and Voronoi deformation density (VDD) analysis of the charge distribution. 相似文献
13.
14.
A Bunker PT Männistö J.-F. St. Pierre T Róg P Pomorski L Stimson 《SAR and QSAR in environmental research》2013,24(1-2):179-189
Results from extensive 70 ns all-atom molecular dynamics simulations of catechol-O-methyltransferase (COMT) enzyme are reported. The simulations were performed with explicit TIP3P water and Mg2?+ ions. Four different crystal structures of COMT, with and without different ligands, were used. These simulations are among the most extensive of their kind and as such served as a stability test for such simulations. On the methodological side we found that the initial energy minimization procedure may be a crucial step: particular hydrogen bonds may break, and this can initiate an irreversible loss of protein structure that becomes observable in longer time scales of the order of tens of nanoseconds. This has important implications for both molecular dynamics and quantum mechanics–molecular mechanics simulations. 相似文献
15.
Yue Lai Xiao Kuang Wen-Hong Yang Yu Wang Ping Zhu Jing-Pu Li Xia Dong Du-Jin Wang 《高分子科学》2021,39(2):154-163
Stimulus-responsive polymers containing dynamic bonds enable fascinating properties of self-healing, recycling and reprocessing due to enhanced relaxation of polymer chain/network with labile linkages. Here, we study the structure and properties of a new type of thermoplastic polyurethanes (TPUs) with trapped dynamic covalent bonds in the hard-phase domain and report the frustrated relaxation of TPUs containing weak dynamic bond andπ-πinteraction in hard segments. As detected by rheometry, the aromatic TPUs with alkyl disulfide in the hard segments possess the maximum network relaxation time in contrast to those without dynamic bonds and alicyclic TPUs. In situ FTIR and small-angle scattering results reveal that the alkyl disulfide facilitates stronger intermolecular interaction and more stable micro-phase morphology inπ-πinteraction based aromatic TPUs. Molecular dynamics simulation for pure hard segments of model molecules verify that the presence of disulfide bonds leads to strongerπ-πstacking of aromatic rings due to both enhanced assembling thermodynamics and kinetics. The enhancedπ-πpacking and micro-phase structure in TPUs further kinetically immobilize the dynamic bond. This kinetically interlocking between the weak dynamic bonds and strong molecular interaction in hard segments leads to much slower network relaxation of TPU. This work provides a new insight in tuning the network relaxation and heat resistance as well as molecular self-assembly in stimulus-responsive dynamic polymers by both molecular design and micro-phase control toward the functional applications of advanced materials. 相似文献
16.
Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins. 相似文献
17.
Jiankuai Diao Andrew J. Maniotis Robert Folberg Emad Tajkhorshid 《Theoretical chemistry accounts》2010,125(3-6):397-405
Fibronectins (FNs) are a major component of the extracellular matrix (ECM), and provide important binding sites for a variety of ligands outside and on the surface of the cell. Similar to other ECM proteins, FNs are consistently subject to mechanical stress in the ECM. Therefore, it is important to study their structure and binding properties under mechanical stress and understand how their binding and mechanical properties might affect each other. Although certain FN modules have been extensively investigated, no simulation studies have been reported for the FN type I (Fn1) domains, despite their prominent role in binding of various protein modules to FN polymers in the ECM. Using equilibrium and steered molecular dynamics simulations, we have studied mechanical properties of Fn1 modules in the presence or the absence of a specific FN-binding peptide (FnBP). We have also investigated how the binding of the FnBP peptide to Fn1 might be affected by tensile force. Despite the presence of disulfide bonds within individual Fn1 modules that are presumed to prevent their extension, it is found that significant internal structural changes within individual modules are induced by the forces applied in our simulations. These internal structural changes result in significant variations in the accessibility of different residues of the Fn1 modules, which affect their exposure, and, thus, the binding properties of the Fn1 modules. Binding of the FnBP appears to reduce the flexibility of the linker region connecting individual Fn1 modules (exhibited in the form of reduced fluctuation and motion of the linker region), both with regard to bending and stretching motions, and hence stabilizes the inter-domain configuration under force. Under large tensile forces, the FnBP peptide unbinds from Fn1. The results suggest that Fn1 modules in FN polymers do contribute to the overall extension caused by force-induced stretching of the polymer in the ECM, and that binding properties of Fn1 modules can be affected by mechanically induced internal protein conformational changes in spite of the presence of disulfide bonds which were presumed to completely abolish the capacity of Fn1 modules to undergo extension in response to external forces. 相似文献
18.
19.
Yen TY Joshi RK Yan H Seto NO Palcic MM Macher BA 《Journal of mass spectrometry : JMS》2000,35(8):990-1002
Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds. 相似文献
20.
One challenge in protein refolding is to dissociate the non-native disulfide bonds and promote the formation of native ones. In this study, we present a coarse-grained off-lattice model protein containing disulfide bonds and simulate disulfide bond shuffling during the folding of this model protein. Introduction of disulfide bonds in the model protein led to enhanced conformational stability but reduced foldability in comparison to counterpart protein without disulfide bonds. The folding trajectory suggested that the model protein retained the two-step folding mechanism in terms of hydrophobic collapse and structural rearrangement. The disulfide bonds located in the hydrophobic core were formed before the collapsing step, while the bonds located on the protein surface were formed during the rearrangement step. While a reductive environment at the initial stage of folding favored the formation of native disulfide bonds in the hydrophobic core, an oxidative environment at a later stage of folding was required for the formation of disulfide bonds at protein surface. Appling a dynamic redox environment, that is, one that changes from reductive to oxidative, intensified disulfide bond shuffling and thus resulted in improved recovery of the native conformation. The above-mentioned simulation was experimentally validated by refolding hen-egg lysozyme at different urea concentrations and oxidized glutathione/reduced glutathione (GSSG/GSH) ratios, and an optimal redox environment, in terms of the GSSG to GSH ratio, was identified. The implementation of a dynamic redox environment by tuning the GSSG/GSH ratio further improved the refolding yield of lysozyme, as predicted by molecular simulation. 相似文献