Protein Stabilization by Introduction of Cross-Strand Disulfides

Abstract

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.

Spackling the Crack: Stabilizing Human Fibroblast Growth Factor-1 by Targeting the N and C terminus β-Strand Interactions

“…Several studies of mutational effects upon protein stability have proposed a significant contribution to overall ΔG from energetic changes in the denatured state; the majority of these describing a reduced entropy of unfolding. [20][21][22] Substitution of Lys12 and Asn95 by β-branched Val may reduce the entropy of the denatured state due to side-chainbackbone interactions unique to β-branched residues, 17,18 thus making a favorable contribution to overall ΔG. However, similar increases in stability are observed at position 12 with Cys mutations (a non-β-branched residue).…”

The Cyclic Cystine Ladder in θ-Defensins Is Important for Structure and Stability, but Not Antibacterial Activity

“…Backbone cyclization and inclusion of disulfide bonds are both established methods for increasing the stability of proteins and peptides (20), making the cyclic cystine ladder a potentially useful scaffold for drug design and protein engineering applications (Fig. 1d).…”

A theoretical study of the stability of disulfide bridges in various β-sheet structures of protein segment models

“…Cystine is more often in NHB than in HB site of proteins [19]. The effect on stability of a cross-strand disulfide within proteins is an ongoing question [19] as disulfide bridges are rarely found between adjacent β strands of proteins. This observation was even once ruled as a 'forbidden' structural scenario [20,21].…”

“…As more information on protein structures accumulate, so have the discovery of more and more cases, where cross-strand disulfides exist [19]. Disulfides also play a role in several other biochemical processes [22][23][24][25][26][27][28].…”