Three Dimensional Structure Determination of Proteins in PERI

Abstract

Ribonuclease F,, the guanine-specific ribonuclease from Fusarium moniliforme(1), was crystallized from 2-methyl-2,4- pentanediol/H,O solution in two different crystal forms, corresponding to RNase F,-2'GMP complex or the inhibitor-free enzyme respectively. The molar ratio of 2'GMP/enzyme in the crystals was determined to be 0.9 by comparing absorbances on UV spectra. The inhibitor-free crystal belongs to orthorhombic space group P2,2,2, with unit cell parameters : a=46.6 A, b-56.3 2, c=31.6 A. The crystal of the complex belongs to hexagonal space group P6, with cell dimensions ; a=b=40.2, c=120.9. The inhibitor-free crystal diffracts X-ray very well beyond 1.5 A and intensity data to 1.8 A were collected with a 4 circle diffractometer ( Enraf-Nonius CAD4 ) on a sealed tube generator. Intensity data were also collected from the complex crystal at 2.3 A resolution. RNase F, is by 59 % homologous in sequence with RNase T, (2) of which three dimensional structure was already determined with respect to the 2'GMP-enzyme complex(3,4). The structure analysis of RNase Fy, was carried out about the inhibitor-free crystal, using molecular replacement technique. We could trace the whole main chain of RNase F,. Although its entire conformation including secondary structure is similar to that of RNase Tj, considerable differences were observed in loop structures. This may reflect the conformational alteration caused by binding to 2'GMP (5).