Department of Chemistry

Dr Itzhaki is now working at the Hutchinson/MRC Research Centre

Our research combines a range of different approaches, including protein engineering, biophysical methods, functional studies. We work closely with molecular and cell biologists and medical geneticists in order to correlate the residue-specific information obtained about the protein architecture to understanding the mechanisms of function of proteins in vivo. Links with theoreticians in all aspects of the proposal will help towards the ultimate goal of predicting protein function and malfunction from amino acid sequence.

We are focussing on three main areas of research:
(1) Structural mechanisms of regulation of the eukaryotic cell cycle and related processes such as transcription and DNA repair. The proper development of multicellular organisms is a complex process that requires precise control of cell proliferation via a network of extracellular and intracellular signaling pathways that process growth regulatory signals. This signaling network is superimposed upon the basic cell cycle machinery that regulates progression through the phases of the cell cycle, such as cell growth, DNA replication, and cell division. The components of the detailed biochemical circuitry controlling the cell cycle are now emerging. A deeper understanding of cell cycle events at the molecular level will require characterisation of the structure, function, and interactions between the component proteins. Ultimately we may be able to simulate the complex cascade of interactions that constitute a signaling pathway. But for this to be possible, we will need quantitative information concerning binding affinities and the conformational changes, domain movements and allosteric effects, that characterises each interaction involved in the process.

(2) Protein folding and function: With more and more sequencing information available, a distinct class of protein structures has begun to emerge. These are composed of tandem repeats of small motifs that stack in a linear fashion to produce elongated structures. The motifs are ubiquitous and involved in a wide range of biological processes. However, it is not yet clear how these small structured motifs carry out their functions. Can they work in isolation or is the total more than the sum of the parts, with multiple motifs providing extended surfaces for molecular recognition? The fundamentally different nature of linear repeat structures compared with globular proteins makes them a novel and important focus for protein folding studies and for testing recent theories that have emerged from studies on globular proteins.

(3) Protein misfolding and disease: a. Aggregated states: Amyloidogenic proteins are thought to unfold partially and then refold to an alternate conformation that enables assembly into fibrils. We are investigating domain swapping, a process also involving breaking apart the structure in order to exchange a domain with a partner molecule, as a potential mechanism for fibril formation. b. Disease-associated mutations: In the past, protein engineering has been used to probe the contribution of individual residues to protein structure by making 'conservative' types of mutations — usually a small truncation of the side chain — within a 'model' protein. We are now ideally placed to quantify the structural effect of non-conservative mutations such as those that cause disease and to interpret the increasing wealth of data on sequence variants that occur in the general population. Using a range of approaches, we are quantifying the effect of disease-causing mutations on structure, stability, solubility and folding. The biological consequences, such as the fate of mutant proteins (aggregation, degradation, location) in the cell, will also be examined.

Selected Publications

1. Seeliger, M. A., Schymkowitz, J. W. H., Rousseau, F., Wilkinson, H. R. and Itzhaki, L. S. Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2. Biochemistry (2001) in press.

2. Schymkowitz, J. W. H., Rousseau, F., Wilkinson, H. R., Friedler, A. and Itzhaki, L. S. Observation of signal transduction in 3D domain swapping. Nature Structural Biology (2001) 8, 888-896.

3. Rousseau, F., Schymkowitz, J. W. H., Wilkinson, H. R. and Itzhaki, L. S. Domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues. Proc. Natl. Acad. Sci. USA (2001) 98, 5596-6001.

4. Schymkowitz, J. W. H., Rousseau, F. and Itzhaki, L .S. Sequence conservation provides the best prediction of the role of proline residues in p13suc1. Journal of Molecular Biology (2000) 301, 201-206.

5. Schymkowitz, J. W. H., Rousseau, F., Irvine, L. R. and Itzhaki, L .S. The folding pathway of the cell-cycle regulatory protein p13suc1: clues for the mechanism of domain swapping. Structure With Folding and Design (2000). 8, 89-100.

6. Tang, K. S., Guralnick, B., Wang, W. K., Fersht, A. R. and Itzhaki, L. S. Stability and folding of the tumour suppressor protein p16. Journal of Molecular Biology (1999) 285, 1869-1886.

7. Clarke, J. and Itzhaki, L. S. Hydrogen exchange and protein folding and stability. Current Opinions in Structural Biology (1998) 8, 112-118.

8. Rousseau, F., Schymkowitz, J. W. H., Sanchez del Pino, M. and Itzhaki, L. S. Stability and folding of the cell cycle regulatory protein, p13suc1. Journal of Molecular Biology (1998) 284, 503-519.