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: 10039, 10040, 10041, 10042, 10043, 10044, 10045, 10046, 10047, 10048, 10049, 10050, 10051, 10052, 10053, 10054, 10055, 10056, 10057, 10058, 10059, 10060, 10061, 10062 Extra Details: alfa-helix; active-site geometry; inhibitor conformation;,thermal stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:39 p.m.
|Number of data points||61|
|Proteins||Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1|
|Assays/Quantities/Protocols||Experimental Assay: dTm temp:25.0 C ; Experimental Assay: dCp ; Experimental Assay: ddG ; Experimental Assay: dTm ; Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT|