| Despite a wealth of biochemical and structural information, the structure of filamentous actin (F-actin) remains to be elucidated. The tendency of monomeric actin to polymerize into filaments of random length precludes crystallization; therefore, models of the filamentous form of actin have been constructed using data obtained by electron microscopy and fiber diffraction of two-dimensional paracrystals as constraints. These models, however, have not definitively fit all the biochemical cross-linking data, and remain open to speculation. Until an atomic-resolution crystal structure of F-actin is determined, the mechanism of motile activity will not be solved.; The first step in achieving a crystal structure of F-actin is the purification of discrete oligomers of actin. This work describes two such approaches utilizing the actin-binding proteins gelsolin and tropomyosin, and a cyclic peptide, phalloidin. In the first, tropomyosin is added to gelsolin:actin filaments at a ratio of 14 actin:2 tropomyosin:1 gelsolin, purified, and crystallized. Structural and biochemical experiments revealed that instead of forming a complex with the desired ratio, tropomyosin caused an annealing into longer filaments. In addition, gelsolin was found to (at least) partially dissociate from the actin filaments, but remains decorated to tropomyosin-stabilized filaments, suggesting hitherto unknown regulatory functions for both tropomyosin and gelsolin.; The second method employs gelsolin and phalloidin. Upon addition of phalloidin to short gelsolin:actin filaments, two chromatographic species are formed. The larger of these is a heterogeneous population of complexes with an average of fourteen actin per gelsolin, containing phalloidin. The second species is a homogeneous complex comprised of three actin per gelsolin, but devoid of phalloidin. This complex can be concentrated and re-chromatographed, and is the first known discrete oligomer of defined length to be purified. This species is a promising candidate for crystallization, and can reveal intrafilament contacts, as well as the structural interactions between gelsolin and actin. |
