X‐ray crystallographic and calorimetric studies of the effects of the mutation Trp59→ Tyr in ribonuclease T1

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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, P21, with two molecules/asymmetric unit, W59Y: a= 4.934 nm, b= 4.820 nm, c= 4.025 nm, β= 90.29°. Y45W/W59Y: a= 4.915 nm, b= 4.815 nm, c= 4.015 nm, β= 90.35°. 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 α‐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.