Structure, Dynamics and Function of Actin

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Actin, a major cellular protein, plays a key role in motion and force generation in muscle and non-muscle cells. The regulation of actin's diverse cellular functions involves specific interactions with many actin-binding proteins. These interactions depend on the equilibration of actin monomers (G-actin) and filaments (F-actin) among different conformational states. The objective of this research is to understand the dynamics and structural states of actin and their role in the actin-based processes. The specific aims of this project include: (i) mapping the intermolecular interfaces in F-actin and the dynamic changes at these interfaces; (ii) elucidating the functional role of the interface dynamics; (iii) determining the structural basis of F-actin severing by cofilin, a major cellular actin remodeling protein; and (iv) testing the model of the ADP/ATP dependent switch between the open and closed nucleotide cleft conformations of the actin monomer. Investigations of the functional role of interface dynamics will include measurements of: (i) interface perturbation by actin binding proteins, and (ii) assays of changes in motion generation with myosin and in the interaction with other proteins due to cross-linking constraints on the interface dynamics. These aims will be pursued through multidisciplinary and collaborative studies (using wild type and mutant proteins with cysteinyl residues engineered for labeling with fluorescence and spin probes) involving cross-linking, electron microscopy, fluorescence, mass spectrometry, electron paramagnetic resonance, kinetics, and single molecule measurements.The broader impact of this project will be in advancing the discovery and understanding of basic cellular processes while providing interdisciplinary training in the chemistry/biology interface to students at all levels - including summer trainees from underrepresented groups at the local colleges - and preparing them for academic and industrial careers.