Prof. Neil Isaacs Department of Chemistry University of Glasgow Glasgow G12 8QQ Scotland Telephone: +44 (0) 141 330 5954 FAX: +44 (0) 330 4888 email: neil@chem.gla.ac.uk

My research is concerned with the determination of protein structures using single crystal X-ray diffraction methods in order to understand the biological function of the protein.

Glycoprotein hormones: Human chorionic gonadotropin (hCG) is one of a family of structurally related glycoprotein hormones involved in the control of reproductive processes. The structure of the hormone was determined in 1994 (see A.J. Lapthorn entry). Work is continuing to extend the resolution of the structure and to map the antigenic surface of the molecule by determining the structures of a number of complexes formed between hCG and Fab fragments from monoclonal antibodies.

Bacterial light-harvesting complexes: In collaboration with Professor Cogdell (Institute of Biomedical and Life Sciences, IBLS) the structure of a light-harvesting (LH2) complex from the photosynthetic bacterium Rhodopseudomonas acidophila was determined in 1995 (see A.A. Freer entry). The resolution of this structure is being extended and neutron experiments will be undertaken at the Institute Laue- Langevin (Grenoble) to probe the protein/lipid/detergent interface in the complex and crystal. Natural light-harvesting complexes with different light absorption characteristics are being purified and crystallised to study how these properties are modulated by the protein. Mutants, designed to probe the structure/function relationship of the LH2 complex, are being prepared in collaboration with Professor Hunter at Sheffield.

Mutant reaction centres from photosynthetic bacteria: In collaboration with Professors Richard Cogdell (IBLS), Neil Hunter (Sheffield) and Dr. Mike Jones (Sheffield), mutants of the photosynthetic reaction centre from Rhodobacter sphaeroides, designed to have altered functional properties, have been prepared and crystallised. The determination of the structures is in progress. An analysis of the structure will lead to a more complete understanding of the role of the proteins in the function of the reaction centre.

Macrophage inhibitory protein MIP-1a: MIP-1a is a small protein (Mr 8,000) which inhibits the proliferation of haemopoeitic stem cells. This property makes it of potential use in cancer therapies. By allowing the immune system to be shut down, more concentrated anti-cancer therapies can be used without the risk of damaging the immune system. Although it is a small protein, MIP-1a readily forms large aggregates, making it difficult to study in vitro. By mutating charged residues on the surface of the molecule, mutants have been produced which are either monomeric, dimeric or tetrameric. In collaboration with Dr. Graham (Beatson Institute) crystals of each of these forms have been grown and the structures determined. Refinement of the structures is in progress.

Type II dehydroquinase from mycobacterium tuberculosis: This enzyme catalyses the dehydration of 3-dehydroquinate to 3-dehydroshikimate, a reaction common to both the biosynthetic shikimate pathway and the catabolic quinate pathway. There are two classes of dehydroquinase with different mechanisms: the type I enzymes catalyse a syn (cis) elimination while the type II enzymes catalyse an anti (trans) elimination. The mechanism of the type II enzyme is unknown. In collaboration with Professor Coggins (IBLS) the structure of the dehydroquinase from Mycobacterium tuberculosis has been determined and is presently being refined. The structure may allow for the design of a novel inhibitor which will have potential interest in the treatment of tuberculosis.

P69 pertactin from bordetella pertussis: Bordetella pertussis is the causative agent of whooping cough and P69 pertactin is the acellular domain of an outer membrane 90Kd protein. This protein is a virulence factor and is responsible for binding to host cells. A new generation of acellular whooping cough vaccines contains P69 pertactin as a major component. The structure of the protein has a b-helix fold with the RGD (Arg-Gly-Asp) tripeptide motif implicated in cell adhesion at the beginning of a loop extending from the b-helix. In collaboration with Drs. Charles (UCL) and Fairweather (Glaxo-Wellcome) this work is continuing in order to improve the resolution of the structure; to study the structure of an RGE mutant which lacks cell adhesion properties; and to crystallise a complex between P69 pertactin and its immunodominant epitope.

The C-terminal fragment of tetanus toxin: Tetanus toxin and the seven antigenically distinct forms of botulinum toxin show 27-51% amino acid sequence similarity. Each neurotoxin is sunthesised as a single polypeptide chain of 150,000 Mr, and undergoes proteolytic cleavage to produce a di-chain toxin consisting of the N-terminal 50,000 Mr fragment (light chain) linked by a disulphide bond to the 100,000 Mr carboxy terminal fragment (heavy chain). In the case of tetanus toxin, fragment C (the C- terminal half of the heavy chain) binds to nerve cells. The N-terminal half then forms channels to allow the translocation of the light chain across the cell membrane. Once inside the cell, the light chain inhibits the release of neurotransmitters causing spastic paralysis. We have crystallised the C-terminal fragment of tetanus toxin and are determining the crystal structure in collaboration with Drs. Fairweather (Glaxo- Wellcome) and Charles (UCL).