We propose to systematically develop Laue diffraction methods for fast time-resolution imaging of enzyme catalysis in protein crystals in real time. We will study the reaction catalyzed by bovine pancreatic ribonuclease (Rnase A) in the crystalline state as a test system because it is a standard enzyme that has been studied in many pioneering experiments in molecular biology and its kinetic mechanism is therefore relatively well understood. This project will improve our ability to understand many aspects of fast processes in biology that relate to phenomena in human health. Crystalline Rnase A is catalytically active, catalyzing the hydrolysis of 3'-5' phosphodiester bonds in single-stranded RNA. Crystalline Rnase A remains active in aqueous alcohol solutions at low temperatures, and its reaction rate is easily modulated by temperature. By selecting a proper temperature for the system, we can therefore choose a reaction rate in these crystals that is consistent with the speed of our data collection methods. We will introduce substrate into Rnase A crystals at -65 degrees C in a flow cell, then initiate the reaction by quickly raising the temperature with an infrared laser. We will carry out these studies at the National Synchrotron Light Source on an x-ray beam with a wide energy bandwidth. The resulting diffraction image, called a Laue pattern, captures a large fraction of the Bragg spots for this crystal. We will re-record this same Laue pattern many times sequentially during the time course of a single reaction event within the crystal. Changes in the Bragg intensities that occur during the reaction will then yield information about kinetic changes occurring in the enzyme structure during the reaction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM044292-01
Application #
3303451
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1991-01-01
Project End
1993-12-31
Budget Start
1991-01-01
Budget End
1991-12-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Organized Research Units
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637