This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Catechol O-methyltransferase (COMT) catalyzes critical steps in the metabolism of catechol-estrogens and the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Approximately 25% of the U.S. Caucasian population is homozygous for a valine-to-methionine polymorphism at residue 108 of COMT, which is located in a surface loop ~16 from the active site. Despite the seemingly innocuous position of the polymorphism, 108M COMT loses activity much more rapidly than 108V COMT at physiological temperatures. CD and fluorescence measurements show that the TM of 108M COMT is ~5 C lower than that of the 108V protein. Molecular dynamics simulations indicate that 108M COMT exists in a broader ensemble of potentially deformed conformational states at 37 C than the wildtype protein. The 108M allele has been associated with increased risk for breast cancer, alcoholism, and aggressive and highly antisocial manifestations of schizophrenia. Interestingly, 108M COMT has also been linked to increased sensitivity to pain, and improvement in prefrontal cognitive function, especially working memory. We seek to identify the structural and dynamic differences between the 108V and 108M proteins, and to determine how these differences contribute to the decreased stability of 108M COMT. NMR is a powerful technique that facilitates the study of both protein structure and dynamics in solution. We plan to use NMR to determine the solution structure of both COMT proteins, and to map out how the introduction of a methionine at position 108 affects residue dynamics throughout the protein. We have identified buffer conditions in which both proteins remain soluble and stable for several weeks at suitably high concentrations. Preliminary experiments show 190 of 210 non-proline backbone amides resolved in HSQC-TROSY spectra of both COMT proteins. The HSQC spectra show multiple peak shifts between the 108V and 108M proteins, suggesting that the two solution structures do differ. Specific 15N-labeling of leucine and lysine residues show that these structural differences extend throughout the protein, and are not limited to the region immediately surrounding residue 108. TROSY-based triple resonance spectra are needed both to determine the solution structure of COMT and to identify the specific residues affected by the V108M polymorphism.
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