The isolation of N-acetyl- l-tryptophanyl-α-chymotrypsin

Abstract

Although conformational changes are essential features of proteins, the conformational basis of protein activity is not yet understood at the molecular and atomic levels. X-Ray studies could reveal the mechanism of enzyme action at the atomic level, if they could provide information about the structure of proteins during the course of their reactions. While new data acquisition techniques under development may significantly reduce the time and increase, the precision of recording diffraction patterns, it is obvious that X-ray diffraction techniques will be restricted to the study of conformations and intermediates, which are stable for periods that exceed the normal half-life of these transient species. It is therefore necessary to increase the lifetime of such species such that their three-dimensional structures may be determined in the same manner as a native enzyme. This chapter deals with the temporal resolution of enzyme reactions normally occurring in a few seconds or minutes, but which are sufficiently reduced in rate by lowering the temperature to permit enzyme- substrate intermediates to be stabilized in fluid mixed solvents. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, are also described. Limitations and perspectives of the procedure are discussed critically.