A Nearly Isosteric Photosensitive Amide-Backbone Substitution Allows Enzyme Activity Switching in Ribonuclease S

摘要

ψ[CS-NH]~4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala~4-Ala~5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. ψ[CS-NH]~4-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2′,3′-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of ψ[CS-NH]~4-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t~(1/2) = 180 s) permitted us to determine the enzymatic activity of cis ψ[CS-NH]~4-RNase S by measurement of inital rates of cCMP hydrolysis. Despite thermodynamic stability of cis ψ[CS-NH]~4-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.