Principal Investigator David Ollis Project s06
Research School of Chemistry Machine VP
Co-Investigators Paul Carr, Karen Edwards, Eong Cheah, Yibin Xu and Anna Robinson,
Research School of Chemistry
Protein Refinement and Engineering
We have determined the structures of a number of proteins and need to improve the accuracy of our present models by least squares refinement. In this work we refine our models against the observed x-ray diffraction data with restraints placed on the geometry of the protein model.
Project 1 PII Protein
Bacteria are capable of using ammonia as their sole source of nitrogen. The chemistry required to do this is rather complex and needs to be varied depending on the level of ammonia in the environment. We have solved the structure of a key signal transducing protein in the regulatory cascade that regulates the metabolic incorporation of ammonia.
What are the basic questions addressed?
In previous years we have obtained the structure of the PII protein. Recent biochemical studies have shown that PII interacts with both ATP and alpha ketoglutarate and this ternary complex that is of biological importance.
What are the results to date and future of the work?
The structure of the protein with ATP was obtained by soaking ATP into existing crystals of the protein. Apart from the location of the ATP binding site, substantial changes in the structure of the protein were observed. The atomic refinement of the complex structure has been completed and a publication describing this work is now being prepared.
A new crystal form of the protein with both ATP and
alpha ketoglutarate has been obtained and used to collect x-ray
diffraction data. A partial model of the protein has been used
in molecular replacement calculations to locate the molecule in
the new cell. X-plor is now being used to refine the structure
of the ternary complex.
Project 2 Dienelactone Hydrolase
Dienelactone hydrolase (DLH) is an enzyme used by bacteria and fungi in the degradation of aromatic compounds. It is a monomeric protein of 25,000 Daltons that catalyses the hydrolysis of a lactone. The structure of the protein was determined using x-ray crystallography and was found to be an alpha/beta protein. The active site contains a catalytic triad of three residues that are linked by H-bonds in a manner similar to that first found in the serine protease chymotrypsin.
What are the basic questions being addressed?
We would like to understand how DLH interacts with substrates and inhibitors.
What are the results to date and the future of the work?
We have obtained crystals of DLH with bound inhibitor. This complex forms crystals that are quite different to the native. The structure of the complex was obtained using molecular replacement methods and is currently being refined prior to publication.
What computational techniques are used and why is a supercomputer required?
We use a non-linear least squares method as implemented in the program X-PLOR. This method of protein refinement reduces the amount of human intervention required. The X-PLOR program would not run efficiently on the workstations in the Research School of Chemistry.
A Theoretical Study of Substrate Induced Activation
of Dienelactone Hydrolase, A. J. Beveridge,
and D.L.Ollis, Protein Engineering, (1995) 8 (2) 135-142.
Crystal Structure of the Escherichia coli Quininone
Oxidoreductase Complexed with NAD P. J. M Thorn, J.D. Barton,
N.E Dixon, D.L.Ollis, & K.J Edwards, . J. Mol. Biol, 249
X-Ray Structure of the Signal Transducing Protein
PII from Escherichia coli
P.D. Carr, U.E. Cheah, P.M. Suffolk, S.G. Vasudevan, N.E. Dixon,
& D.L. Ollis, ACTA Cryst D, D52, 93-104.(1996)
Crystallization and Preliminary X-ray Diffraction
Studies of New Crystal Ligand Forms of Escherichia coli PII
Complexed with Various Ligands. K.J. Edwards,
P.M. Suffolk, P.D. Carr, M. Wegman, E Cheah, and D.L Ollis, submitted.