Conformational analysis and design of disulfides in antiparallel β‐sheets and helices

Abstract

A nonredundant data set having 5025 polypeptides with 2311 disulfides was used to study cross‐strand disulfides (CSDs) in antiparallel beta‐sheets, and intrahelical disulfides (IHDs).75 and 1 CSDs were found at non‐hydrogen‐bonded (NHB) and hydrogen‐bonded (HB) registered pairs, respectively. Disulfides at HB pairs lead to steric repulsions with main chain and require a rare, positive χ1 value for at least one of the Cys. 13 disulfides were introduced in NHB and HB pairs in 4 model proteins: Leu binding protein (LBP), Leu, Ile, Val binding protein (LIVBP), maltose binding protein (MBP), and Top7. The LIVBP mutant did not show disulfide formation. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the exposed NHB LBP mutant showed an increase of 3.1 °C in Tm. Both exposed and 2 of the 3 buried NHB Top7 mutants were stabilized. All 4 HB Top7 mutants were destabilized (ΔΔGo = −3.3 to −6.7 kcal/mol). Thus, CSDs engineered at exposed NHB pairs can be used to improve protein stability.35 examples were found of IHDs between the N‐Cap and 3rd helical positions. Cys pairs were introduced at N‐Cap‐3; 1,4; 7,10 in 2 helices of an E. coli thioredoxin (Trx) mutant. Disulfides spontaneously formed only at positions N‐Cap‐3 during purification. All oxidized and reduced mutants were destabilized relative to nSS Trx. All mutants were mildly redox active.