X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in ribonuclease T1.


Abstract

Two mutants of ribonuclease T1 (RNaseT1), [59-tyrosine]ribonuclease T1 (W59Y) and [45-tryptophan,59-tyrosine]ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the inhibitor 2'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P2(1), with two molecules/asymmetric unit, W59Y: a = 4.934 nm, b = 4.820 nm, c = 4.025 nm, beta = 90.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, beta = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single alpha-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]ribonuclease T1 (Y24W), [42-tryptophan]ribonuclease T1 (Y42W), [45-tryptophan]ribonuclease T1 (Y45W), [92-alanine]ribonuclease T1 (H92A) and [92-threonine]ribonuclease T1 (H92T) with and without the further mutation Trp59-->Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability. Study holds ProTherm entries: 12010, 12011, 12012, 12013, 12014, 12015, 12016, 12017, 12018, 12019, 12020, 12021 Extra Details: alfa-helix; active-site geometry; inhibitor conformation;,thermal stability

Submission Details

ID: k8Sv6DJ33

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Schubert WD;Schluckebier G;Backmann J;Granzin J;Kisker C;Choe HW;Hahn U;Pfeil W;Saenger W,Eur. J. Biochem. (1994) X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in ribonuclease T1. PMID:8125111
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