Synchrotron x-radiation crystallographic studies of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus

Abstract

The synchrotron X-radiation crystallographic study of the enzyme 6-phosphogluconate dehydrogenase (6-phospho-D gluconate NADP+ oxidoreductase (decarboxylating), EC 1.1.1.44) extracted from Bacillus stearothermophilus is reported. This crystallographic data was unobtainable using conventional sources. During this project I was involved in the building and commissioning of the central facility for protein crystallography at the SRS, Daresbury, (Helliwell et al (1982)). Chapter Three gives a detailed description of the instrument along with the relevant theory required for calculating beam compression, Guiner position and spectral resolution. Further expressions for optimising the beam geometry are derived from first principles. Chapter Four describes the data collection from bacterial 6PGDH crystals and details the data reduction procedures used to obtain a final dataset of integrated intensities using the MOSCO suite of programs. There is also a discussion of radiation damage in protein crystals within this chapter.
Chapter One gives background information on the bacterial 6PGDH enzyme including its preparation and properties. It also discusses thermal stability in microorganisms, concentrating particularly on proteins from the thermophilic bacteria. In addition, there is a discussion of the relationships between the various dehydrogenase structures that have to date been solved. A summary of the relevant diffraction theory applicable to protein crystallography is given in Chapter Two.
The technique of molecular replacement was used in an attempt to obtain initial phases for the bacterial data. The theory of the method and the results of our calculations are given in Chapters Five and Six. The molecular replacement calculations revealed a direction of the non crvstallocrraohic dimer axis and also allowed us to determine the correct space group as P3221. The thesis is concluded with some closing remarks and suggestions for further work.