Physical Studies of Actin Nucleation and Conformational Dynamics

Actin is a 42 kilodalton ATPase that exists ubiquitously in eukaryotic cells. Unlike other ATPases, however, actin, under suitable conditions, can polymerize, forming helical filaments. Cells, in orchestrating their myriad cellular processes, utilize actin's intrinsic capacity to polymerize, but do so in a tightly controlled fashion, such that new filaments only appear when and where the cell needs them to suit specific purposes. Such control exists at two different levels. Firstly, the stability of actin filaments is subject to "intrinsic" control arising from the state of bound nucleotide. ATP binding favors incorporation of actin monomers into filaments. This incorporation augments actin's ATP hydrolysis activity, and the conversion of ATP to ADP in the nucleotide binding cleft considerably destabilizes filaments, facilitating the return of filament subunits to free monomers. The structural mechanism through which nucleotide conveys information throughout the actin monomer to influence polymerization behavior remains poorly understood and represents a persistent fundamental biological question. In this work I, for the first time, apply modern muti-resonance NMR methods to begin to answer these questions. In addition to the aforementioned intrinsic control, cellular actin is subject to "extrinsic" control via the action of nucleation factors. In order to form a growing filament, actin must proceed through a nucleation step in which monomers must assemble into a thermodynamically and kinetically disfavored nucleus, which ultimately proceeds to a growing filament. Nucleation factors accelerate the rate of filament formation by binding to actin monomers and arranging them into the prerequisite nucleus. In this work, I reveal the crystal structure of actin monomers in complex with the bacterially derived nucleation factor, VopL. The structure represents the first high resolution snapshot of a filament-like nucleation intermediate, and reveals general principles underlying the action of nucleation factors.