COMPARISON OF HYDROGEN-EXCHANGE RATES FOR BOVINE PANCREATIC TRYPSIN-INHIBITOR IN CRYSTALS AND IN SOLUTION

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by H-1 NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35-degrees-C, and pH 9.4 and 25-degrees-C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or tum. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule. Backbone atoms of Ile 18 and Tyr 35 are mutually hydrogen bonded to each other at the open end of a pair of twisted antiparallel beta-sheet strands, which are closed at the other end in a hairpin-like arrangement by a short turn. Ile 18 and Tyr 35 exchange rates are slowed by 4-5 orders of magnitude in crystals. Their location in the protein suggests that crystallization selectively damps motions of the open end of the beta-sheet, which connects the flexible loops with the more rigid central core of the molecule.