Structural investigation of gelsolin superfamily members

We expressed three recombinant proteins in E. coli and purified them for analysis. Human gelsolin fragment G2-G3 retains the ability of intact gelsolin to bind to and co-sediment with filamentous actin. Tryptophan fluorescence intensity from G2-G3 is Ca²⁺-sensitive, increasing with Ca²⁺ concentration in two steps, suggesting that the binding of Ca²⁺ at two sites induces a structural change that reduces the solvent exposure of tryptophan side chains. Iodide quenching studies of tryptophan fluorescence from the fragment indicate reduced effectiveness of the quencher when G2-G3 is in the presence of excess free Ca²⁺, consistent with the above conclusion. Screening of crystallization conditions for G2-G3 on its own and in complexes with actin yielded only small crystals of G2-G3 that did not diffract X-rays sufficiently well to yield structural information. Human CapG possesses three domains, C1-C3, that closely resemble the three N-terminal domains of gelsolin. We show CapG to exhibit both actin filament nucleation and severing activities in light scattering and total internal reflection fluorescence assays. Crystallization screens yielded crystals of CapG suitable for X-ray diffraction analysis, which resulted in a 1.5 Å resolution model for the C2-C3 fragment of CapG. The structure reveals a very different packing of C2-C3 in comparison to that observed previously in a mutant, CapG-sev, that had been engineered to resemble gelsolin G1 in portions of C1 and in the linking peptide between C1 and C2 [Zhang et al. (2006) EMBO J 25, 4458-4467]. We conclude that these structures represent analogues of the two different activated forms of the N-terminal half of gelsolin bound to actin [Wang (2008) Ph.D. thesis, UBC]. Finally, human villin-6M consists of villin domains V4-V6 without headpiece, which closely resemble gelsolin G4-G6. Screening of crystallization conditions produced crystals from which X-ray diffraction data generated a 1.9 Å resolution model for villin domain V6. Although grown by a different procedure, the crystals yielded a structure for V6 that is identical to that reported previously [Wang et al.,2009].Science, Faculty ofChemistry, Department ofGraduat