CONFORMATIONAL ANALYSIS OF TRYPSIN AT ATOMIC RESOLUTION REVEALS EFFECTS OF CRYSTALLINE ENVIRONMENT. Hans D. Bartunik, Alexandre N. Popov, Max-Planck Research Unit for Structural Molecular Biology, Protein Dynamics Group, MPG-ASMB c/o DESY, Notkestraße 85, 22603 Hamburg, Germany

The crystalline environment affects the relative orientation of the two ß- barrel domains of bovine pancreatic ß-trypsin (BPT) and induces conformational strain. The comparison is based on anisotropic refinement using SHELXL-93 [1] of BPT at 5deg.C in two different orthorhombic crystal forms [2, 3] at 1.0-1.1 Å resolution (R=8.3%; 9.8%). The accuracy in the atomic positions is on average 0.01-0.02 Å for each structural model. When aligned on the active site region and one domain (150 residues; rmsd 0.09 Å, max. deviation 0.19 Å for the main chain atoms), the other domain exhibits relative coordinate shifts (rmsd 0.22 Å, max. 0.52 Å) as well as substantial changes in the conformational angles. The positional and conformational differences are particularly large for the ß-strands 81-90 (near the surface) and 104-108. Molecular packing interactions further induce flexibility in a number of residues (35 in the one structure, 24 in the other) that are present in discrete alternate conformations. Correlations between alternate side chain locations are observed which extend over distances up to 20 Å; in several cases, water or sulphate molecules with partial occupancies are involved. Most of the ordered solvent (ca. 2.2 waters per accessible residue - nearly all in the first coordination shells) and the degree of anisotropy in the individual atomic temperature factors are essentially not affected by the crystalline environment. In both structures, only one residue (Gln 192) is not located in well-defined electron density; the flexibility may reflect its functional role in orienting substrates. The diffraction data were measured on the synchrotron beamline BW6/DORIS.

[1] Sheldrick, G. W. (1993). SHELXL-93, Univ. Göttingen.

[2] Marquart, M. et al. (1983). Acta Crystallogr. sect. B, 39, 480-490.

[3] Bartunik, H. D. et al. (1989). J. Mol. Biol. 210, 813-828.