Wildemann Dirk, Schiene-Fischer Cordelia, Aumüller Tobias, Bachmann Annett, Kiefhaber Thomas, Lücke Christian, Fischer Gunter
Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle/Saale, Germany.
J Am Chem Soc. 2007 Apr 25;129(16):4910-8. doi: 10.1021/ja069048o. Epub 2007 Mar 31.
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. psiCS-NH-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 psiCS-NH-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 psiCS-NH-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psiCS-NH-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.
psi[CS-NH]4-RNase S是通过在Ala4-Ala5肽键处进行硫代化（O/S交换）获得的RNase S的位点特异性修饰版本，用于评估蛋白质主链光开关对生物活性的影响。psiCS-NH-RNase S是通过S蛋白与相应化学合成的硫代化S肽衍生物重组产生的。与RNase S的比较表明，该复合物具有相似的热力学稳定性，并且对胞苷2',3'-环一磷酸（cCMP）具有未受干扰的酶活性。在紫外线照射条件（254 nm）下，psiCS-NH-RNase S的硫代肽键在光稳态下发生可逆光异构化，顺式/反式异构体比例大幅增加。缓慢的热再异构化（t(1/2) = 180 s）使我们能够通过测量cCMP水解的初始速率来测定顺式psiCS-NH-RNase S的酶活性。尽管顺式psiCS-NH-RNase S具有热力学稳定性，但其酶活性完全丧失，但再异构化后恢复。我们得出结论，硫代肽键修饰的多肽主链是蛋白质定点光开关的通用探针。
