High torsional energy disulfides : relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent β-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained β-sheet, are required.

Cross-strand disulfides in the non-hydrogen bonding site of antiparallel β-sheet (aCSDns): poised for biological switching

Forbidden disulfides are stressed disulfides found in recognisable protein contexts previously defined as structurally forbidden. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. The meta-stability of forbidden disulfides makes them likely candidates as redox switches. Here we mined the Protein Data Bank for examples of the most common forbidden disulfide, the aCSDn. This is a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen bonded moieties are directed away from each other. We grouped these aCSDns into homologous clusters and performed an extensive physicochemical and informatic analysis of the examples found. We estimated their torsional energies using quantum chemical calculations and studied differences between the preferred conformations of the computational model and disulfides found in solved protein structures to understand the interaction between the forces imposed by the disulfide linkage and typical constraints of the surrounding β-sheet. In particular, we assessed the twisting, shearing and buckling of aCSDn-containing β-sheets, as well as the structural and energetic relaxation when hydrogen bonds in the motif are broken. We show the strong preference of aCSDns for the right-handed staple conformation likely arises from its compatibility with the twist, shear and Cα separation of canonical β-sheet. The disulfide can be accommodated with minimal distortion of the sheet, with almost all the strain present as torsional strain within the disulfide itself. For each aCSDn cluster, we summarise the structural and strain data, taxonomic conservation and any evidence of redox activity. aCSDns are known substrates of thioredoxin-like enzymes. This, together with their meta-stability, means they are ideally suited to biological switching roles and are likely to play important roles in the molecular pathways of oxidative stress.

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°, χ390, 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.

Potential role of glutathione in evolution of thiol-based redox signaling sites in proteins.

Cysteine is susceptible to a variety of modifications by reactive oxygen and nitrogen oxide species, including glutathionylation; and when two cysteines are involved, disulfide formation. Glutathione-cysteine adducts may be removed from proteins by glutaredoxin, whereas disulfides may be reduced by thioredoxin. Glutaredoxin is homologous to the disulfide-reducing thioredoxin and shares similar binding modes of the protein substrate. The evolution of these systems is not well characterized. When a single Cys is present in a protein, conjugation of the redox buffer glutathione may induce conformational changes, resulting in a simple redox switch that effects a signaling cascade. If a second cysteine is introduced into the sequence, the potential for disulfide formation exists. In favorable protein contexts, a bistable redox switch may be formed. Because of glutaredoxin's similarities to thioredoxin, the mutated protein may be immediately exapted into the thioredoxin-dependent redox cycle upon addition of the second cysteine. Here we searched for examples of protein substrates where the number of redox-active cysteine residues has changed throughout evolution. We focused on cross-strand disulfides (CSDs), the most common type of forbidden disulfide. We searched for proteins where the CSD is present, absent and also found as a single cysteine in protein orthologs. Three different proteins were selected for detailed study-CD4, ERO1, and AKT. We created phylogenetic trees, examining when the CSD residues were mutated during protein evolution. We posit that the primordial cysteine is likely to be the cysteine of the CSD which undergoes nucleophilic attack by thioredoxin. Thus, a redox-active disulfide may be introduced into a protein structure by stepwise mutation of two residues in the native sequence to Cys. By extension, evolutionary acquisition of structural disulfides in proteins can potentially occur via transition through a redox-active disulfide state.

An analysis of side chain interactions and pair correlations within antiparallel β-sheets : the differences between backbone hydrogen-bonded and non-hydrogen-bonded residue pairs

Cross-strand pair correlations are calculated for residue pairs in antiparallel β-sheet for two cases: pairs whose backbone atoms are hydrogen bonded together (H-bonded site) and pairs which are not (non-H-bonded site). The statistics show that this distinction is important. When glycine is located on the edge of a sheet, it shows a 3:1 preference for the H-bonded site. Thestrongest observed correlations are for pairs of disulfide-bonded cystines, many of which adopt a close-packed conformation with each cystine in a spiral conformation of opposite chirality to its partner. It is likely that these pairs are a signature for the family of small, cystine-rich proteins. Most other strong positive and negative correlations involve charged and polar residues. It appears that electrostatic compatibility is the strongest factor affecting pair correlation. Significant correlations are observed for β- and γ-branched residues inthe non-H-bonded site. An examination of the structures showsa directionality in side chain packing. There is a correlation between (1) the directionality in the packing interactions of non-H-bonded β- and γ-branched residue pairs, (2) the handedness of the observed enantiomers of chiral β-branched side chains, and (3) the handedness of the twist of β-sheet. These findings have implications for the formation of β-sheets during protein folding and the mechanism by which the sheet becomes twisted

Between-strand disulfides: Forbidden disulfides linking adjacent ß-Strands

Between-strand disulfides (BSDs) connect cysteine (Cys) residues across adjacent strands of β-sheets. There are four BSD types which can be found in regular β-structure: CSDs, which link residues immediately opposite each other in the β-structure (residues i and j); ETDs, which connect Cys out of register by one residue (i and j ± 1); BDDs, which join Cys at positions i and j ± 2; and BFDs, which link residues i and j ± 3. Formation of these disulfides was initially predicted to be forbidden, producing too much local strain in the protein fold. However, BSDs do exist in nature. Significantly, their high levels of strain allow them to be involved in redox processes under physiological conditions. Here we characterise BSD motifs found in the Protein Data Bank (PDB), discussing important intrinsic factors, such as the disulfide conformation and torsional strain, and extrinsic factors, such as the influence of the β-sheet environment on the disulfide and vice versa. We also discuss the biological importance of BSDs, including the prevalence of non-homologous examples in the PDB, the conservation of BSD motifs amongst related proteins (BSD clusters) and experimental evidence for BSD redox activity. For clusters of homologous BSDs we present detailed data of the disulfide properties and the variations of these properties amongst the “redundant” structures. Identification of disulfides with the potential to be involved in biological redox processes via the analysis of these data will provide important insights into the function and mechanism of BSD-containing proteins. Characterisation of thiol-based redox signalling pathways will lead to significant breakthroughs in understanding the molecular basis of oxidative stress and associated pathways, such as ageing and neurodegenerative diseases.

Cross cultural exchange to support reasoning about socio-scientific sustainability issues

In this article, we describe a project on reasoning about socio-scientific issues (SSIs), involving French and Australian pre-service science teachers engaged in on-line discussion and development of a wiki. In the research, we developed frameworks for looking at the quality of reasoning about 'socially acute' sustainability questions. We found the level of reasoning was enhanced by the cross-cultural exchange, and identified the importance of context in framing reasoning quality. We argue that science teachers could effectively adapt this approach to develop students' scientific literacy and embed the 'science as a human endeavour' strand of the Australian Curriculum in their practice.