Actin protein engineering and biochemical characterization of polymerization deficient actin filament precursors

Self-association of actin into long actin filaments is a fundamental characteristic of actin. Over 150 unique actin binding proteins having been identified to date, many of which bind actin filaments (F-actin). F-actin is highly dynamic and not amenable to crystallization and therefore no direct, high-resolution, structural data exists for filamentous actin. The goal of this research is to develop short F-actin oligomers to serve as structural platforms for the elucidation of molecular interactions between F-actin and actin filament binding proteins. Any strategy to this end involves crosslinking of actin subunits and modification of the resulting actin oligomers to inhibit their polymerization. The extent of these modifications must be strategically balanced to maintain actin stability and physiological interactions that are integral to F-actin function. A novel technique utilizing tetramethylrhodamine (TMR) as a reporter of actin thermal stability is described. Actin thermal stability depends on the bound nucleotide and cation. We observed a biphasic trend in the T m of TMR-actin with increasing nucleotide concentration, supporting a two-pathway model for actin unfolding, and emphasizing the relationship between inter-domain flexibility and the maintenance of actin stability. To produce F-actin lateral dimers and trimers, the crosslinker, 1,4-N,N'-phenylenebismaleimide was employed and the oligomers were rendered polymerization deficient using a novel mono-ADP-ribosyltransferase called photox. Photox transfers ADP-ribose to monomeric actin at Arg-177 resulting in the inhibition of actin polymerization. The modifications resulted in a moderate reduction in thermal stability. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar to those of wild-type actin. DNase-I binding affinity under low and high ionic strength showed that the oligomers retain a high degree of conformational flexibility. Furthermore, polymer nucleation was rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin. The combined strategy of chemical crosslinking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds. This work forms the foundation for research involving the use of F-actin oligomers as an F-actin structural scaffold.