The association of some forms of cardiomyopathy, deafness, and blindness with defects in myosins, underscores the importance of studying these actin-associated, molecular motors. An understanding of the molecular mechanism of these enzymes can ultimately provide information crucial to the design of rational therapies to avert these diseases. Class I myosins are small, monomeric, mechanoenzymes with a motor domain, which binds actin and nucleotide; a regulatory region to which light chains attach; and a carboxy terminal tail. Class I myosins are widely expressed in mammalian cells and are predicted to mediate important actin-dependent processes such as cell migration and transport of cargo among intracellular compartments. The broad, long-term objective is to understand the enzymatic mechanism of the mammalian myosin-I, MYR 1, and how it relates to cell function. Key observations made in this laboratory have demonstrated the unique mechanochemical properties of MYR 1. MYR 1-actin exhibits a slow, biphasic transient interaction with nucleotide and a two-step powerstroke observed with single-molecule methods. These properties, not seen before for other myosins, presumably reflect the specialized adaptation of MYR 1 for particular cellular functions. The proposed experiments focus on detailing how MYR 1 interacts with actin and nucleotide.
The specific aims are: (i) To investigate the contribution of two specific subdomains to MYR 1's unique mechanochemical properties using mutant proteins. The ability of mutant MYR 1 to interact with actin and nucleotide will be determined with steady and transient state kinetic analyses, in vitro motility assays and single molecule methods. (ii) To determine if MYR 1 undergoes nucleotide-dependent conformational changes using 3D reconstructions from cryo-electron micrographs of MYR 1-actin. These studies will allow for visualization of structural changes in MYR 1 during the myosin powerstroke. (iii) To determine if MYR 1 associates with specific subpopulations of actin filaments and whether specific structural elements in the motor domain modulate its binding to microfilaments. The ability of mutant MYR 1 to associate with actin filaments in complex with other actin-binding proteins will be determined in vitro with actin co-sedimentation assays and in cells using expressed MYR 1. These studies will provide insight into how motors are targeted to particular sites in the cell, an important question in cell biology. ? ?
