Calcium-binding Proteins
Projects focus on the three-dimensional
structures of calcium-binding proteins and their roles in health and disease.
We use physical biochemical techniques such as high resolution NMR spectroscopy,
analytical centrifugation, fluorescence and circular dichroism to probe
structure/function relationships.
We are particularly interested in a group of proteins called the S100
proteins. These are dimeric signaling molecules belonging to the EF-hand
calcium binding protein family. The proteins undergo a calcium-induced
conformational change and interact with specific target proteins paralleling
the mechanisms of the calcium sensor proteins troponin-C, calmodulin and
recoverin. Recently we uncovered the conformational change in the protein
S100B using multi-dimensional NMR spectroscopy. Currently we are probing
the details of this structural change for S100B and other signalling proteins.
Protein Interactions in Degradation and Parkinson's Disease
The process of ubiquitylation is one of the most important regulatory pathways in all cells. It involves the transfer of ubiquitin (Ub) between a series of proteins until it labels a target protein as a polyubiquitin chain. During the course of ubiquitylation, Ub first forms a high-energy thiolester intermediate with an active site cysteine of the activating enzyme E1 in an ATP-dependent step. The ubiquitin molecule is then transferred to the catalytic cysteine of an E2 conjugating enzyme (Ubc1, Hip2, UbcH7, UbcH8), forming a second thiolester. Labeling of a substrate protein occurs through transfer of the Ub directly from the E2 as in the case of RING E3 enzymes (cCbl, parkin) or through an intermediary transfer to a HECT domain E3. This mechanism is further complicated by a plethora of ubiquitin-like proteins (e.g. SUMO, NEDD8) and ubiquitin-binding domains (e.g. UBA, CUE, UIM) that have accessory functions such as targeting an ubiquitin complex to the proteasome.
Our lab is concentrating on the structures and mechanisms of Ub-E2-E3 complexes in order to define the molecular basis for ubiquitin chain elongation. We have determined the three-dimensional structure of Ubc1 (24 kDa), the first structure for a class II ubiquitin conjugating protein. We are now using our experience with this system to determine how the E2 enzymes Hip2, UbcH7, UbcH8 and Ubc1 interact with ubiquitin, and the E3 ligase proteins cCbl and parkin, enroute to polyubiquitin chain formation.
