? Anesthetic effects on cellular activity manifest as changes in protein function, which might result from direct anesthetic-protein interactions. It is difficult to correlate anesthetic effects on protein function with effects on protein structure and dynamics, in part because there is a dearth of structures of anesthetic-protein complexes. Anesthetics and other hydrophobic drugs are known to affect the ability of calmodulin (CaM) to bind ligands and activate enzymes. The binding of volatile anesthetics to Calmodulin (CaM) is Ca2+- dependent, which indicates they bind to locations that are either not present or not accessible in apoCaM. This makes a compelling argument for determining the structure of an anesthetic-CaM complex, because the structure would reveal the component(s) of Ca2+-CaM structure specific to anesthetic binding. In this project, two specific aims are proposed: A) determine the structure of a volatile anesthetic-CaM complex and B) determine how volatile anesthetics affect CaM function.
In specific aim A, NMR methods will be used to estimate the Kd of an anesthetic-CaM complex; determine the structure of an anesthetic-CaM complex, and; determine the effects of anesthetic on the dynamics of CaM. This work will use NMR methods to determine . structure because the Ca2+-CaM resonances have already been assigned, which will expedite the process, and NMR methods are able to elucidate the effects of anesthetics on CaM dynamics.
In specific aim B, the effects of anesthetic binding on the Ca2+ affinity of CaM, the binding of peptides to CAM, and the ability of CaM to activate enzymes will be studied with standard biochemical techniques. When this project is completed, it will have demonstrated that volatile anesthetics bind to CaM under specific conditions, determined the solution structure of a volatile anesthetic-CaM complex, and explained how volatile anesthetics might affect the interaction of CaM with proteins. ? ?
