Molecular Dynamics Simulations of Proteins with Chemically Modified Disulfide Bonds

Proteins that are used as therapeutic drugs act in the extracellular microenvironment. They usually have a small number of intramolecular disulfide bonds to help maintain their tertiary structure in the vascular circulation. In general, most cysteine residues are part of a disulfide bond with free sulfhydrals being uncommon. We have studied whether the site-specific chemical reduction of disulfides and the incorporation of a 3-carbon methylene bridge between the cysteines in interferon-α 2a would change the structure of this protein. Bridging of both of the disulfide bonds of interferon-α 2a was studied using two different molecular simulation protocols: (1) molecular dynamics, and (2) stochastic dynamics. We have shown that the disulfide bonds in interferon-α 2a can be reduced and chemically modified without significantly altering the tertiary structure of the protein. This offers the novel possibility of chemically modifying therapeutically important proteins without affecting their biological properties.     

Understanding Ramachandran plot for dipeptide: A density functional theory and information-theoretic approach study

The Ramachandran plot in biochemistry visualizes the propensity of amino acid residues to assume different secondary structures. It can be obtained by different approaches such as bioinformatics and molecular dynamics. In this work, we systematically scan the two single bonds around the α-carbon of a peptide to generate the three-dimensional potential energy surface and then project it onto the coordinate plane to generate the Ramachandran plot. Using the two schemes of the total energy partition and information-theoretic approach in density functional theory, we analyze the plot and find out that the dominant contributor to determine the local minima of secondary structures, especially β-sheets, in the Ramachandran plot is the electrostatic interaction, whereas steric and exchange-correlation contributions play minor yet indispensable roles, especially for determining α-helixes. As the generalization of our prior studies for systems with one rotatable bond, our current results confirm what we yielded before from the viewpoint of energetic contributions, and at meanwhile provide an in-depth understanding about the nature and origin of secondary structure propensities from the Ramachandran plot.     

Deciphering structural and functional roles of disulfide bonds in decorsin

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.     

Redox-dependent formation of disulfide bonds in human replication protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)(4)-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H(2)O(2) treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA.     

Thiol‐disulfide redox equilibria of glutathione metaboloma compounds investigated by tandem mass spectrometry

The thiol group of cysteine plays a pivotal role in structural and functional biology. We use mass spectrometry to study glutathione‐related homo‐ and heterodimeric disulfides, aiming at understanding the factors affecting the redox potentials of different disulfide/thiol pairs. Several electrospray ionization (ESI)‐protonated disulfides of cysteamine, cysteine, penicillamine, N‐acetylcysteine, N‐acetylpenicillamine, γGluCySH, HSCyGly, and glutathione were analyzed on a triple quadrupole instrument to measure their energy‐resolved tandem mass spectra. Fission of the disulfide bond yields RSH*H⁺ and RS⁺ ions. The logarithm of the intensity ratio of the RS⁺/RSH*H⁺ fragments in homodimeric disulfides is proportional to the normal reduction potential of their RSSR/RSH pairs determined by nuclear magnetic resonance (NMR) in solution, the more reducing ones yielding the higher ratios. Also in some R₁S‐SR₂ disulfides, the ratio of the intensities of the RSH + H⁺ and RS⁺ ions of each participating thiol shows a linear relationship with the Nernst equation potential difference of the corresponding redox pairs. This behavior allows us to measure the redox potentials of some disulfide/thiol pairs by using different thiol‐reducing probes of known oxidoreductive potential as reference. To assist understanding of the fission mechanism of the disulfide bond, the fragments tentatively identified as ‘sulfenium’ were themselves fragmented; accurate mass measurement of the resulting second‐generation fragments demonstrated a loss of thioformaldehyde, thus supporting the assigned structure of this elusive intermediate of the oxidative stress pathway. Understanding this fragmentation process allows us to employ this technique with larger molecules to measure by mass spectrometry the micro‐redox properties of different disulfide bonds in peptides with catalytic and signaling biological activity. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization of the disulfides of bio-thiols by electrospray ionization and triple-quadrupole tandem mass spectrometry

Glutathione and other intracellular low molecular mass thiols act both as the major endogenous antioxidant and redox buffer system and, as recently highlighted, as an important regulator of cellular homeostasis. Such cellular functions are mediated by protein thiolation, a newly recognized post-translational modification which involves the formation of mixed disulfides between GSH and key disulfide-linked Cys residues in the native protein structure. It is also well known that thiol-seeking heavy metals, such as mercury, cadmium and lead, may interfere in this regulatory system, thus disrupting the cellular functioning. To identify such mixed disulfides in order to investigate their biological role, 15 homo- and heterodimeric disulfides were prepared by air oxidation of binary mixtures containing cysteine, homocysteine, penicillamine, N-acetylcysteine, N-acetylpenicillamine and glutathione and their protonated molecules were characterized by mass spectrometry. Collisionally activated unimolecular decomposition of protonated homo- and heterodimeric disulfides generated by electrospray ionization gives rise to fission of the disulfide system both between the two sulfur atoms and across the C--S bonds, to yield structurally specific fragments which allow one to define the structure of the compounds and to discriminate between isomeric compounds. Fission between the sulfur atoms yields a pair of R--S(+) ions and, in some cases, also the complementary fragments corresponding to the protonated amino acids. Fission across the C--S bonds mainly occurs in the disulfides of N-acetylcysteine and N-acetylpenicillamine and gives rise to non-S-containing fragments formally similar to those obtained from some mercapturic acids. The complementary fragments, formally connected as R--S--S(+) ions are also observed. Fragmentation of glutathione disulfides mainly shows the characteristic loss of the terminal gamma-linked glutamic acid and little, if any, fragmentation of the disulfide system.     

Synthesis, redox properties, and conformational analysis of vicinal disulfide ring mimics

A vicinal disulfide ring (VDR) results from disulfide-bond formation between two adjacent cysteine residues. This eight-membered ring is a rare motif in protein structures and is functionally important to those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR mimics, discernment of the preferred conformation of these mimics, and the determination of their respective disulfide redox potentials.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

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.     

Multiple Disulfide-Bonded States of Native Proteins: Estimate of Number Using Probabilities of Disulfide Bond Formation

The polypeptide backbone of proteins is held together by two main types of covalent bonds: the peptide bonds that link the amino acid residues and the disulfide bonds that link pairs of cysteine amino acids. Disulfide bonds form as a protein folds in the cell and formation was assumed to be complete when the mature protein emerges. This is not the case for some secreted human blood proteins. The blood clotting protein, fibrinogen, and the protease inhibitor, α2-macroglobulin, exist in multiple disulfide-bonded or covalent states in the circulation. Thousands of different states are predicted assuming no dependencies on disulfide bond formation. In this study, probabilities for disulfide bond formation are employed to estimate numbers of covalent states of a model polypeptide with reference to α2-macroglobulin. When disulfide formation is interdependent in a protein, the number of covalent states is greatly reduced. Theoretical estimates of the number of states will aid the conceptual and experimental challenges of investigating multiple disulfide-bonded states of a protein.     

Improving the accuracy of predicting disulfide connectivity by feature selection

Disulfide bonds are primary covalent cross‐links formed between two cysteine residues in the same or different protein polypeptide chains, which play important roles in the folding and stability of proteins. However, computational prediction of disulfide connectivity directly from protein primary sequences is challenging due to the nonlocal nature of disulfide bonds in the context of sequences, and the number of possible disulfide patterns grows exponentially when the number of cysteine residues increases. In the previous studies, disulfide connectivity prediction was usually performed in high‐dimensional feature space, which can cause a variety of problems in statistical learning, such as the dimension disaster, overfitting, and feature redundancy. In this study, we propose an efficient feature selection technique for analyzing the importance of each feature component. On the basis of this approach, we selected the most important features for predicting the connectivity pattern of intra‐chain disulfide bonds. Our results have shown that the high‐dimensional features contain redundant information, and the prediction performance can be further improved when these high‐dimensional features are reduced to a lower but more compact dimensional space. Our results also indicate that the global protein features contribute little to the formation and prediction of disulfide bonds, while the local sequential and structural information play important roles. All these findings provide important insights for structural studies of disulfide‐rich proteins. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry

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.     

Effects of aromatic thiols on thiol-disulfide interchange reactions that occur during protein folding

The folding of disulfide containing proteins from denatured protein to native protein involves numerous thiol-disulfide interchange reactions. Many of these reactions include a redox buffer, which is a mixture of a thiol (RSH) and the corresponding disulfide (RSSR). The relationship between the structure of RSH and its efficacy in folding proteins in vitro has been investigated only to a limited extent. Reported herein are the effects of aliphatic and especially aromatic thiols on reactions that occur during protein folding. Aromatic thiols may be particularly efficacious as their thiol pK(a) values and reactivities match those of the in vivo catalyst, protein disulfide isomerase (PDI). This investigation correlates the thiol pK(a) values of aromatic thiols with their reactivities toward small molecule disulfides and the protein insulin. The thiol pK(a) values of nine para-substituted aromatic thiols were measured; a Hammett plot constructed using sigma(p-) values yielded rho = -1.6 +/- 0.1. The reactivities of aromatic and aliphatic thiols with 2-pyridyldithioethanol (2-PDE), a small molecule disulfide, were determined. A plot of reactivity versus pK(a) of the aromatic thiols had a slope (beta) of 0.9. The ability of these thiols to reduce (unfold) the protein insulin correlates strongly with their ability to reduce 2-PDE. Since the reduction of protein disulfides occurs during protein folding to remove mismatched disulfides, aromatic thiols with high pK(a) values are expected to increase the rate not only of protein unfolding but protein folding as well.     

Novel multi-epitope protein containing conserved epitopes from different Leishmania species as potential vaccine candidate: Integrated immunoinformatics and molecular dynamics approach

Leishmaniosis, caused by intracellular parasites of the genus Leishmania, has become a serious public health problem around the world, and for which there are currently extensive limitations. In this work, a theoretical model was proposed for the development of a multi-epitope vaccine. The protein GP63 of the parasite was selected for epitopes prediction, due to its important biological role for the infection process and abundance. IEDB tools were used to determine epitopes B and T in Leishmania braziliensis; besides, other conserved epitopes in three species were selected. To improve immunogenicity, 50S ribosomal protein L7 / L12 (ID: P9WHE3) was used as a domain of adjuvant in the assembly process. The folding arrangement of the vaccine was obtained through homologous modeling multi-template with MODELLER v9.21, and a Ramachandran plot analysis was done. Furthermore, physicochemical properties were described with the ProtParam tool and secondary structure prediction combining GOR-IV and SOPMA tools. Finally, a molecular dynamics simulation (50 ns) was performed to establish flexibility and conformational changes. The analysis of the results indicates high conservancy in the epitopes predicted among the four species. Moreover, Ramachandran plot, physicochemical parameters, and secondary structure prediction suggest a stable conformation of the vaccine, after a minimum conformational change that was evaluated with the free energy landscape. The conformational change does not drive any substantial change for epitope exposition on the surface. The vaccine proposed could be tested experimentally to guide new approaches in the development of pan-vaccines; vaccines with regions conserved in multiple species.     

diSBPred: A machine learning based approach for disulfide bond prediction

The protein disulfide bond is a covalent bond that forms during post-translational modification by the oxidation of a pair of cysteines. In protein, the disulfide bond is the most frequent covalent link between amino acids after the peptide bond. It plays a significant role in three-dimensional (3D) ab initio protein structure prediction (aiPSP), stabilizing protein conformation, post-translational modification, and protein folding. In aiPSP, the location of disulfide bonds can strongly reduce the conformational space searching by imposing geometrical constraints. Existing experimental techniques for the determination of disulfide bonds are time-consuming and expensive. Thus, developing sequence-based computational methods for disulfide bond prediction becomes indispensable. This study proposed a stacking-based machine learning approach for disulfide bond prediction (diSBPred). Various useful sequence and structure-based features are extracted for effective training, including conservation profile, residue solvent accessibility, torsion angle flexibility, disorder probability, a sequential distance between cysteines, and more. The prediction of disulfide bonds is carried out in two stages: first, individual cysteines are predicted as either bonding or non-bonding; second, the cysteine-pairs are predicted as either bonding or non-bonding by including the results from cysteine bonding prediction as a feature.The examination of the relevance of the features employed in this study and the features utilized in the existing nearest neighbor algorithm (NNA) method shows that the features used in this study improve about 7.39 % in jackknife validation balanced accuracy. Moreover, for individual cysteine bonding prediction and cysteine-pair bonding prediction, diSBPred provides a 10-fold cross-validation balanced accuracy of 82.29 % and 94.20 %, respectively. Altogether, our predictor achieves an improvement of 43.25 % based on balanced accuracy compared to the existing NNA based approach. Thus, diSBPred can be utilized to annotate the cysteine bonding residues of protein sequences whose structures are unknown as well as improve the accuracy of the aiPSP method, which can further aid in experimental studies of the disulfide bond and structure determination.     

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER.     

Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.     

Site-specific protein modification by genetic encoded disulfide compatible thiols

Cysteine chemistry provides a low cost and convenient way for site-specific protein modification.However,recombinant expression of disulfide bonding containing protein with unpaired cysteine is technically challenging and the resulting protein often suffers from significantly reduced yield and activity.Here we used genetic code expansion technique to introduce a surface exposed self-paired dithiol functional group into proteins,which can be selectively reduced to afford active thiols.Two compounds containing self-paired disulfides were synthesized,and their genetic incorporations were validated using green fluorescent proteins(GFP).The compatibility of these self-paired di-thiols with natural disulfide bond was demonstrated using antibody fragment to afford site-specifically labeled antibody.This work provides another valuable building block into the chemical tool-box for site-specific labeling of proteins containing internal disulfides.