MECHANISM OF PROMISCUOUS INHIBITION BY SMALL MOLECULE AGGREGATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The scientific community has long been troubled by the presence of nonspecific small molecules that inhibit proteins other than their hypothesized targets. The behavior of a large and diverse subset of these inhibitors can be explained by a common mechanism: aggregation. At micromolar concentrations some nonspecific molecules form large aggregates that inhibit a wide range of unrelated soluble proteins. From previous studies we know that inhibition results from the direct interaction of protein and aggregates. This interaction is reversible and does not result in denaturation, but the extent of our understanding stops there. How does binding to the aggregate lead to inhibition? Does the aggregate bind to a particular protein surface or is it nonspecific? Does binding restrain or induce dynamic motion in the protein? I will use deuterium exchange mass spectrometry to address these questions.Since we know that aggregates induce changes subtler than denaturation, it is necessary that we use a more sensitive technique. Mass spectrometry coupled to deuterium exchange of amide hydrogens provides evidence of how exposed certain regions are to solvent. This allows us to examine the accessibility of surface protons and the overall dynamics of the enzyme through the exchange of core (or buried) protons. We predict that aggregates inhibit by altering the normal dynamic motions of the enzyme. Additionally, we will be able to determine whether there is a particular surface to which the aggregate binds. There are few techniques that allow this level of sensitivity and among them mass spectrometry is the most practical. These studies will provide insight into the mechanism of promiscuous inhibition by aggregation, thus elucidating a novel interaction between small molecules and their biological targets.