A nearly isosteric photosensitive amide-backbone substitution allows enzyme activity switching in ribonuclease s.
psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[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 psi[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 psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[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.
Wildemann
Dirk
D
Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Schiene-Fischer
Cordelia
C
Aumüller
Tobias
T
Bachmann
Annett
A
Kiefhaber
Thomas
T
Lücke
Christian
C
Fischer
Gunter
G
D000577:Amides / Q000737:chemistry; D003563:Cyclic CMP / Q000737:chemistry; D006868:Hydrolysis; D007536:Isomerism; D010777:Photochemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000528:radiation effects*; D013816:Thermodynamics; D014466:Ultraviolet Rays
D000577:Amides; D003563:Cyclic CMP; C520930:RNase S, psi(CS-NH)(4)-; D012260:Ribonucleases; C022542:ribonuclease S