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

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• 作者：Dirk Wildemann ; Cordelia Schiene-Fischer ; Tobias Aumü ; ller ; Annett Bachmann ; Thomas Kiefhaber ; Christian Lü ; cke ; Gunter Fischer
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：April 25, 2007
• 年：2007
• 卷：129
• 期：16
• 页码：4910 - 4918
• 全文大小：187K
• 年卷期：v.129,no.16(April 25, 2007)
• ISSN：1520-5126

文摘

[CS-NH]⁴-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/Sexchange) at the Ala⁴-Ala⁵- peptide bond, was used to evaluate the impact of protein backbonephotoswitching on bioactivity. [CS-NH]⁴-RNase S was yielded by recombination of the S-protein and therespective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealedsimilar 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 ratioof the thioxopeptide bond of [CS-NH]⁴-RNase S in the photostationary state occurred under UV irradiationconditions (254 nm). The slow thermal reisomerization (t^1/2 = 180 s) permitted us to determine the enzymaticactivity of cis [CS-NH]⁴-RNase S by measurement of inital rates of cCMP hydrolysis. Despitethermodynamic stability of cis [CS-NH]⁴-RNase S, its enzymatic activity is completely abolished butrecovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbonerepresents a versatile probe for site-directed photoswitching of proteins.