Protein stabilization by introduction of cross-strand disulfides.
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.
Chakraborty
Kausik
K
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Thakurela
Sudhir
S
Prajapati
Ravindra Singh
RS
Indu
S
S
Ali
P Shaik Syed
PS
Ramakrishnan
C
C
Varadarajan
Raghavan
R
D000818:Animals; D004220:Disulfides / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D029968:Escherichia coli Proteins; D013879:Thioredoxins