Enhanced stability of human prion proteins with two disulfide bridges

We compare the folding equilibrium of the globular domain of the human prion protein with two variants of this domain, for which an additional disulfide bond was introduced into the location where it is found in the naturally occurring doppel protein. We find that the unfolding transition midpoint of the variants is shifted toward higher denaturant concentration, indicating that the engineered disulfide bond significantly stabilizes the global protein structure. Our results further reveal that the two-disulfide variant proteins, while possessing the same global fold as the wild-type, display marked differences in their folding pathway-in particular, the absence of a characteristic alpha-helix to beta-sheet transition, which is a fundamental feature associated with misfolding of proteins into amyloid fibrils, especially in the context of prion diseases. These surprising characteristics of disulfide mutant prion proteins have important implications for the understanding of the generic aberrant processes leading to amyloid fibril formation and protein aggregation, as well as providing insight into possible therapeutic strategies.     

Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ₂, χ₃, χ₂′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ₂～ ? 60°, χ₃～90, χ₂′～ ? 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.     

A method for covalent insertion of mercury into the cysteine disulfide bridges of proteins

A method is described by which atomic mercury can be taken up by thiol groups and inserted into the disulfide bridges of proteins which can be reversibly reduced and denatured. The method utilizes tandem columns of Sephadex G-10 and Biogel P2. Protein samples are separated from reducing and denaturing agent on the Sephadex column and then react with mercury, which is bound to the Biogel P2 column. Of eight proteins tested, all took up mercury using this method. The amount of mercury incorporated by this method differed from that found using other methods and was closer to the stoichiometry of the disulfide bridges of the protein than these methods.     

Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis

A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with C alpha-C alpha distances of less than or equal to 6.5 A and C beta-C beta distances of less than or equal to 4.5 A are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S-S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at less than or equal to 2 A resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.     

Interchain disulfide bridges in ribonuclease BS-1

RNAase BS-1, a dimeric ribonuclease isolated from bovine seminal plasma, is made up of two identical subunits whose amino acid sequence is homologous to the sequence of bovine pancreatic RNAase A. The dimeric structure, resistant to denaturating agents, is sensitive to thiol reagents even in the absence of denaturants. The isolation and characterization of a cystine peptide containing two adjacent $\text{1}\text{2}\text{cystine}$ residues is reported. As the peptide molecular weight is halved after reductive cleavage with dithiothreitol, a structure based on two interchain disulfide bonds between the two adjacent $\text{1}\text{2}\text{cystine}$ of each subunit is proposed. The singularity of such a structure for a small enzymatic protein is discussed.     

Scorpion venom peptides without disulfide bridges

Several hundred disulfide-bridged neurotoxic peptides have been characterized from scorpion venom; however, only few scorpion venom peptides without disulfide bridges have been identified and characterized. These non-disulfide-bridged peptides (NDBPs) are a novel class of molecules because of their unique antimicrobial, immunological or cellular signaling activities. This review provides an overview of their structural simplicity, precursor processing, biological activities and evolution, and sheds insight into their potential clinical and agricultural applications. Based on their pharmacological activities and peptide size similarity, we have classified these peptides into six subfamilies.     

MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. AVAILABILITY: Freely available at http://metaldetector.dsi.unifi.it. SUPPLEMENTARY INFORMATION: Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.     

Conformations of folded proteins in restricted spaces

A new method is presented to examine the complete range of folded topologies accessible in the compact state of globular proteins. The procedure is to generate all conformations, with volume exclusion, upon a lattice in a space restricted to the individual protein's known compact conformational space. Using one lattice point per residue, we find 10(2)-10(4) possible compact conformations for the five small globular proteins studied. Subsequently, these conformations are evaluated in terms of residue-specific, pairwise contact energies that favor nonbonded, hydrophobic interactions. Native structures for the five proteins are always found within the best 2% of all conformers generated. This novel method is simple and general and can be used to determine a small group of most favorable overall arrangements for the folding of specific amino acid sequences within a restricted space.     

Sterically hindered disulfide bridges in cystine diketopiperazine, cysteinyl-cysteine disulfide and derivatives.

L-Cystine diketopiperazine (1), L-cysteinyl-cysteine disulfide -HCl (2), L-cysteinyl-cysteine disulfide methyl ester -HCl (3), and t-butyloxycarbonyl-L-cysteinyl-cysteine disulfide methyl ester (4) are investigated by CD, ultraviolet, 13C NMR, infrared and laser Raman spectroscopy. The temperature dependence of the 13C NMR signals of 1 reveals an exceptionally high energy barrier of deltaGNo. = 15.8 +/- 0.2 kcal/mol for the reversible change in helicity of the inherently dissymmetric disulfide bridge of 1. The P-helical diastereomer predominates in dimethyl-sulfoxide at 25 degrees C, with 80-85% of the molecules having this configuration. The Cotton effects of 1 are larger and show smaller temperature coefficients than the conformationally more mobile cystine compounds 2 and 3. After dissolving crystals of 1 in 95% ethanol there is a time-dependent decrease of the ellipticity of the negative Cotton effect at 225 nm, indicating a conformational change in going from crystal to solution. Besides 1, 2 and 3 are at present the only known examples of cystine derivatives with C-S-S-C torsional angles around 90 degrees, which do not exhibit optical activity in the long wavelength disulfide absorption, as is predicted for 1 from the Linderberg-Michl model. At 305 nm a new weak Cotton effect was discovered for 1. The solvent dependence of the CD spectra is discussed and the infrared and Raman spectra are assigned.     

The disulfide bridges of duck egg-white lysozyme II

The disposition of the disulfide bonds of duck egg-white lysozyme II was established in detail by the study of cystine containing peptides obtained by enzymic digestions. A comparison of some optical properties of duck II and duck III lysozymes suggests the same disposition of the disulfide bonds in both enzymes.     

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.     

On the correlation of S-S and C-S raman bands frequencies and intensities with conformations of disulfide bridges in proteins

We have studied dependence of the S-S and C-S raman bands frequencies and intensities on the conformation of disulfide bridges. These correlations have been analyzed on the basis of the normal coordinates treatment in the valence force field and from comparison of the raman and X-ray data for the model compounds and proteins. The found regularity of the intensity variations with conformation make it possible to distinguish these spectral perturbations from those associated with the change in the number of disulfide bridges in proteins. Particularly, this was demonstrated on the gamma-irradiated insulin solutions spectra. We also present some data appropriate for estimation of the content of disulfide bridges in proteins using internal intensity standards.