Crystallization of multiple forms of bovine seminal ribonuclease in the liganded and unliganded state

Bovine seminal ribonuclease (BS-RNase) is an intriguing homodimeric enzyme which exists as two conformational isomers, characterized by distinct catal ytic and biological properties, referred to as M x M and M = M. Reduction o f inter-chain disulfide bridges produces a stable monomeric derivative (M) which is still active. This paper reports the screening and optimization of crystallization conditions for growing single diffraction-quality crystals for the various BS-RNase forms. The crystallization trials were performed using both the vapor diffusion and microbatch methods. The M x M dimer was crystallized in the free form from polyethylene glycol (PEG) 4000 at pH 8.5 and as a complex with the substrate analog uridylyl(2'-5')guanosine (UpG) from an unbuffered ammonium sulfate (AS) solution. These two crystal types diffract X-rays to 2.5 and 1.9 Angstrom resolution, respectively. Two diffe rent crystal types were obtained both for the M = M dimer and for the monom eric derivative. (M = M)a crystals, grown from PEG 4000 (8% w/v) at pH 5.6, diffract X-rays to 4.0 Angstrom. At higher PEG concentration (15% w/v) a d ifferent crystal type was obtained, (M = M)b, which showed a better diffrac tion limit (2.5 Angstrom). For the monomer, type (M)a and (M)b crystals, di ffracting X-rays to 2.5 a resolution, were obtained from AS at pH 6.5 and f rom PEG 4000 at pH 8.5, respectively. A comparison with previously crystall ized forms of the dimer M x M and its complexes with uridylyl(2'-5')adenosi ne and 2'-deoxycytidylyl(3'-5')-2'-deoxyadenosine is also presented. The th ree-dimensional structure analysis of (M x M)UpG and (M = M)b is in progres s. (C) 1999 Elsevier Science B.V. All rights reserved.