Amino acid function relates to its embedded protein microenvironment: A study on disulfide‐bridged cystine

In our previous study, we have shown that the microenvironments around conserved amino acids are also conserved in protein families (Bandyopadhyay and Mehler, Proteins 2008; 72:646–659). In this study, we have hypothesized that amino acids perform similar functions when embedded in a certain type of protein microenvironment. We have tested this hypothesis on the microenvironments around disulfide‐bridged cysteines from high‐resolution protein crystal structures. Although such cystines mainly play structural role in proteins, in certain enzymes they participate in catalysis and redox reactions. We have performed and report a functional annotation of enzymatically active cystines to their respective microenvironments. Three protein microenvironment clusters were identified: (i) buried‐hydrophobic, (ii) exposed‐hydrophilic, and (iii) buried‐hydrophilic. The buried‐hydrophobic cluster encompasses a small group of 22 redox‐active cystines, mostly in alpha‐helical conformations in a –C‐x‐x‐C‐ motif from the Oxido‐reductase enzyme class. All these cystines have high strain energy and near identical microenvironments. Most of the active cystines in hydrolase enzyme class belong to buried hydrophilic microenvironment cluster. In total there are 34 half‐cystines detected in buried hydrophilic cluster from hydrolases, as a part of enzyme active site. Even within the buried hydrophilic cluster, there is clear separation of active half‐cystines between surface exposed part of the protein and protein interior. Half‐cystines toward the surface exposed region are higher in number compared to those in protein interior. Apart from cystines at the active sites of the enzymes, many more half‐cystines were detected in buried hydrophilic cluster those are part of the microenvironment of enzyme active sites. However, no active half‐cystines were detected in extremely hydrophilic microenvironment cluster, that is, exposed hydrophilic cluster, indicating that total exposure of cystine toward the solvent is not favored for enzymatic reactions. Although half‐cystines in exposed‐hydrophilic clusters occasionally stabilize enzyme active sites, as a part of their microenvironments. Analysis performed in this work revealed that cystines as a part of active sites in specific enzyme families or folds share very similar protein microenvironment regions, despite of their dissimilarity in protein sequences and position specific sequence conservations. Proteins 2016; 84:1576–1589. © 2016 Wiley Periodicals, Inc.     

Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.     

Reduction of Canine Plasminogen Leads to an Expanded Molecule which Precipitates

Canine plasminogen is made up of seven domains. In each domain there are several cysteines that are linked by disulfide bonds. Reduction of a limited number of the cystines destabilizes the protein such that it precipitates. The bond or bonds that are broken provide about 14 kcal of stabilization energy. Circular dichroism and dynamic light scattering indicate that there is probably an intermediate that is formed prior to precipitation and that the intermediate is somewhat larger than the compact form of plasminogen.     

Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents.

We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.     

Isolation and characterization of lecithin-cholesterol acyltransferase from hog plasma

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified from hog plasma by a highly efficient procedure. The final enzyme preparation was purified 30,000-fold over the starting material and was homogeneous as indicated by polyacrylamide gel electrophoreses in the presence of both SDS and urea. The purified hog lecithin-cholesterol acyltransferase had an apparent molecular weight of 66 000 on SDS-polyacrylamide gel electrophoresis and HPLC and was found to contain about 21.4% (w/w) carbohydrate-hexose, 11.3%; hexosamine, 1.9%; sialic acid, 8.2%. The amino acid composition analysis showed that hog lecithin-cholesterol acyltransferase contains four half cystines per mol; two cysteines were titrated at neutral pH with 5,5'-dithiobis(2-nitrobenzoic acid). Nearly all the phenolic groups were unavailable to the solvent at neutral pH, while they become exposed at around pH 11. Hog lecithin-cholesterol acyltransferase was found to be associated with HDL in the plasma and it prefers HDL as a substrate. The physicochemical properties of hog lecithin-cholesterol acyltransferase were generally similar to those of the human and the rat enzyme.     

Identification of Zinc-ligated Cysteine Residues Based on 13Cα and 13Cβ Chemical Shift Data

Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom’s chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped ¹³Cβ chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding ¹³Cα chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 ¹³Cα/¹³Cβ shift pairs from 79 proteins with known three-dimensional structures, including 86 ¹³Cα and¹³Cβ shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statisical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron–sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn ligands, we analyzed the Protein Data Bank and found that 78% of the Zn ligands are histidine and cysteine (with nearly identical frequencies), and 18% are acidic residues aspartate and glutamate. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Amino-acid sequences of two basic chymotrypsin inhibitors from silkworm larval hemolymph

Amino-acid sequences of two basic chymotrypsin inhibitors from silkworm hemolymph (SCI-I and SCI-II) are determined. They are composed of each 62 amino-acid residues with differences in only two positions to each other. They both contain six half cystines in a similar arrangement as that of Kunitz-type proteinase inhibitor, except for the one amino-acid insertion in the first cysteine frame. The inhibitory activity of SCI-II against trypsin should be attributed to Lys44 displacing Gln44 in SCI-I which has no antitryptic activity.     

Disulphide bridges in globular proteins

Disulphide bridges in proteins of known sequence, connectivity and structure were studied to search for common features. Their distribution, topology, conformation and conservation were analysed in detail. Several general patterns emerge which to some extent dictate disulphide bridge formation. For example, there is a strong preference for shorter connections, with half-cystines separated by less than 24 residues in 49% of all disulphides. Right- and left-handed disulphides occur equally; the left-handed structures adopt one predominant conformation (symmetric χ₁ = ?60 °, χ₂ = ?80 °, χ₃ = t-90 °). Cystines are generally very well conserved, in contrast to cysteines, with a free —SH group, which mutate rapidly. If a disulphide is not conserved, both cystines are mutated. The role of disulphide bridges in globular proteins is discussed.     

Kinetics in the pre-steady state of the formation of cystines in ribonucleoside diphosphate reductase: evidence for an asymmetric complex

Two folded polypeptides, designated R1 and R2, respectively, combine in an as yet undefined stoichiometry to form ribonucleoside diphosphate reductase (ribonucleotide reductase) from Escherichia coli. Two pairs of cysteines in each R1 protomer have been implicated in the enzymatic mechanism. One pair, cysteines 225 and 462, is located in the active site of the enzyme and forms a cystine concomitant with the reduction of the ribonucleotide. The other pair, cysteines 754 and 759, is located near the carboxy terminus and is thought to reduce the cystine in the active site by disulfide interchange; either thioredoxin or glutaredoxin is then thought to reduce the cystine that results. Rapid quenching and site-directed immunochemistry have been used to follow the formation of the cystine in the active site and the peripheral cystine simultaneously during the pre-steady state. Prereduced R1 dimer of ribonucleoside diphosphate reductase, in the presence of ATP and CDP, was mixed with R2 dimer in an apparatus for quench flow. The reaction was quenched with a solution of acetic acid and N-ethylmaleimide, the protein was then precipitated with trichloroacetic acid, and the precipitate was separated into two portions. The percent of the cystine in the active site in one of the portions was determined as described previously [Erickson, H. K. (2000) Biochemistry 39, 9241-9250]. A similar method was employed to determine the percent of the peripheral cystine in the other portion of the precipitate. It was found that while the formation of both of these cystines was initiated by the addition of R2 dimer, presumably as products of the reduction of CDP, the peripheral cystine appeared to form more rapidly and in a higher yield than the cystine in the active site. These results demonstrate that the formation of the cystine between cysteines 754 and 759 of ribonucleotide reductase from E. coli is kinetically competent. A mechanism consistent with the prior formation of the cystine between cysteine 225 and cystene 462 as well as the kinetics for the formation of each cystine with time is presented. Because twice as much of the peripheral cystine than cystine in the active site had formed during the pre-steady state, it follows that the enzymatically competent complex between R1 dimers and R2 dimers cannot be symmetric.     

A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. The function of these cysteines and the significance of their unique location in yeast PDI have been unclear. Previous data (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786) suggest that the internal cysteines exist as a disulfide in the endoplasmic reticulum of Saccharomyces cerevisiae. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.     

Properties of a hydrophobin isolated from the mycoparasitic fungus Verticillium fungicola

Verticillium fungicola, isolated from Agaricus bisporus (commercial mushroom), produced significant extracellular hydrophobin when grown for 7 days in a static liquid culture of synthetic minimal medium. The hydrophobin was purified by precipitation with ammonium sulphate (80% saturation), Sephadex G-100 gel filtration, and hydroxyapatite column chromatography. The purified protein yielded a single band in polyacrylamide gel electrophoresis under native conditions, with an apparent molecular mass of 70 +/- 4 kDa, and also another single band in SDS-PAGE, with a molecular mass of 7 +/- 3 kDa. Molecular mass determined with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) resulted in 7563.9 m/z. The same protein was extracted from the V. fungicola mycelium. Analysis of the amino acid composition revealed the presence of about 50% hydrophobic residues, detecting at least six cysteines, evaluated as cystines, and no free sulfhydryl groups. The protein did not show any glycosylation. On the basis of similarities in hydropathy patterns and solubility characteristics, V. fungicola hydrophobin can be included as a new member of Class II hydrophobins.     

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa.

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.     

马氏钳蝎毒昆虫类神经毒素的分离、纯化及性质研究

经Sephadex G-50,sp-Sephadex C-25二步柱层析法,从山东马氏钳蝎(Bu-thus martensii Karch)粗毒中分离出四种对美洲(虫非)蠊有强直麻痹反应的毒性蛋白组份。其中二个组分在SDS聚丙烯酰胺电泳和等电聚焦电泳上均呈现单一区带,命名为BmK IT-Ⅰ,BmK IT-Ⅱ其pI分别为8.2和8.4,分子量分别为8400和7560。同时还分析了二个组份的氨基酸组成。经DABITC/PITC双偶合法测定了BmKIT-Ⅰ和BmK IT-Ⅱ的N端部份氨基酸排列顺序,它们分别为H_2NVal.Arg.Asp.Ala……H_2NVal.Arg.Asp.Gly……。 电生理学研究表明,纯化的BmK IT-I(1×10~(-5)g/ml)对(虫非)蠊腹Ⅵ神经节的突触传递有阻断作用,阻断后用生理溶液洗,则突触传递可恢复。从同一蝎毒粗毒中分离纯化的哺乳动物类神经毒素BmKⅢ在浓度高出100倍(1×10~(-3)g/ml)时也可以阻断(虫非)蠊腹Ⅵ神经节的突触传递,但用生理溶液冲洗没有观察到恢复。     

In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer

The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.     

Papillomavirus Assembly Requires Trimerization of the Major Capsid Protein by Disulfides between Two Highly Conserved Cysteines

We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids.     

7-Iron ferredoxin revisited

The crystal structure of the 7Fe ferredoxin from Azotobacter vinelandii has been redetermined using area detector data to 2.7-A resolution and a new derivative. Tetragonal crystals of the protein were maintained at pH 8.0. The results show that the structure previously reported was in error and confirms a recent independent report of the structure (Stout, G.H., Turley, S., Sieker, L. C., and Jensen, L. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, in press). The protein fold is similar to the homologous 8Fe ferredoxin structure for the N-terminal half of the protein; the C-terminal residues wrap around this structure. The structure contains a 3Fe cluster coordinated by cysteines 8, 16, and 49 and a 4Fe cluster coordinated by cysteines 20, 39, 42, and 45. However, there are two free sulfhydryls, cysteines 11 and 24, in the new model. Cysteine 24 is in contact with the [4Fe-4S] cluster. Cysteine 11 is shielded from solvent by residues 86-90.     

Structure of a bacterial sensory receptor. A site-directed sulfhydryl study

Cysteines are substituted at six positions in the aspartate receptor, and these mutant proteins are used to investigate three major facets of receptor structure. 1) The surface of the receptor is examined through measurement of the rate constants for chemical modification of the cysteines by aqueous reagents. Different positions exhibit a range of accessibility (for example, Cys-128 most exposed, Cys-36 most buried). 2) The transmembrane structure of the receptor is determined by reaction of the cysteines with a membrane-impermeant reagent. 3) The spatial proximities in the folded structure of specific pairs of cysteines are investigated by disulfide bond formation. These studies illustrate the usefulness of site-directed sulfhydryl chemistry in the analysis of protein structure.     

Amino acid neighbours and detailed conformational analysis of cysteines in proteins.

Here we present an investigation of the contacts that cysteines make with residues in their three-dimensional environment and a comprehensive analysis of the conformational features of 351 disulphide bridges in 131 non-homologous single-chain protein structures. Upstream half-cystines preferentially have downstream neighbours, whereas downstream half-cystines have mainly upstream neighbours. Non-disulphide bridged cysteines (free cysteines) have no preference for upstream or downstream neighbours. Free cysteines have more contacts to non-polar residues and fewer contacts to polar/charged residues than half-cystines, which correlates with our observation that free cysteines are more buried than half-cystines. Free cysteines prefer to be located in alpha-helices while no clear preference is observed for half-cystines. Histidine and methionine are preferentially seen nearby free cysteines. Tryptophan is found preferentially nearby half-cystines. We have merged sequential and spatial information, and highly interesting novel patterns have been discovered. The number of cysteines per protein is typically an even number, peaking at four. The number of residues separating two half-cystines is preferentially 11 and 16. Left-handed and right-handed disulphide bridges display different conformational parameters. Here we present side chain torsion angle information based on a 5-12 times larger number of disulphide bridges than has previously been published. Considering the importance of cysteines for maintaining the 3D-structural scaffold of proteins, it is essential to have as accurate information as possible concerning the packing and conformational preferences. The present work may provide key information for engineering the protein environment around cysteines.     

Mapping of structural determinants for the oligomerization of p58, a lectin-like protein of the intermediate compartment and cis-Golgi.

Shortly after synthesis, p58, the rat homologue of the mannose-binding lectin ERGIC-53/MR60, which localizes to pre-Golgi and cis-Golgi compartments, forms dimers and hexamers, after which an equilibrium of both forms is reached. Mature p58, a type I membrane protein, contains four cysteine residues in the lumenal domain which are capable of forming disulphide bonds. The membrane-proximal half of the lumenal domain consists of four predicted alpha-helical domains, one heavily charged and three amphipathic in nature, all candidates for electrostatic or coiled-coil interactions. Using single-stranded mutagenesis, the cysteines were individually changed to alanines and the contribution of each of the alpha-helical domains was probed by internal deletions. The N-terminal cysteine to alanine mutants, C198A and C238A and the double mutant, C198/238A, oligomerized like the wild-type protein. The two membrane-proximal cysteines were found to be necessary for the oligomerization of p58. Mutants lacking one of the membrane proximal cysteines, either C473A or C482A, were unable to form hexamers, while dimers were formed normally. The C473/482A double mutant formed only monomers. Deletion of any of the individual alpha-helical domains had no effect on oligomerization. The dimeric and hexameric forms bound equally well to D-mannose. The dimeric and monomeric mutants displayed a cellular distribution similar to the wild-type protein, indicating that the oligomerization status played a minimal role in maintaining the subcellular distribution of p58.