Crystallization of a Complex between Ribonuclease T1 and 2'-Guanylic Acid

Abstract

Ribonuclease TI was crystallized under various conditions. Form I crystals were produced by microdialysis against 53%(v/v) 2‐methyl‐2,4‐pentanediol in 0.01 M sodium acetate, 0.05% 2′‐guanylic acid (2′GMP) and 0.02% NaN₃ (pH 6.2–7.2). These crystals are tetragonal, space group P4₁2₁2 and contain two molecules per asymmetric unit; cell dimensions are a=b= 5.86 nm, c= 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01–0.05 M sodium acetate, 0.25–0.5%, 2′GMP, 0.02% NaN₃ and 2–5 mM calcium acetate (pH 4.0–4.4) in the presence of 50–75% (v/v) 2‐methyl‐2,4‐pentanediol. These crystals are orthorhombic, space group P2₁2₁2₁, and contain one molecule per asymmetric unit; cell dimensions are a= 4.66 nm, b= 5.02 nm, c= 4.04 nm (form I) and a= 4.44 nm, b= 5.00 nm, c= 4.03 nm (form II). Using high‐performance liquid chromatography, it could be shown for all crystal forms that 2′‐GMP is bound in the crystals. The molecular ratio between RNase T₁ and 2′GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme‐nucleotide complex. Heavy‐atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three‐dimensional X‐ray analysis of the RNase‐T₁· 2′GMP complex is under way.