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Structural studies of gelsolin and binding partners

University of British Columbia

Gelsolin is composed of six similarly folded domains, G1 through G6. It controls actin dynamics through its calcium-sensitive ability to bind to, sever, and cap F-actin filaments. This project was designed to test and extend current models for gelsolin activity by growing crystals of intact gelsolin or large fragments of gelsolin in protein complexes with its main target, actin, as well as with specialized gelsolin-specific antibody fragments, nanobodies. Gelsolin-specific nanobodies (GsnVHH) may serve to lock in either resting or activated forms of gelsolin structure, but are of intrinsic interest on their own in that they have potential therapeutic value. This thesis describes cloning, expression, purification, activity assays, crystallization details, and solution of the structures of six proteins and protein complexes. 1) Recombinant human G1-G3 bound to natural source rabbit actin (G1-G3:actin). The present structure is to a higher resolution than earlier ones, providing increased confidence in positioning of amino acid side chains and bound Ca²⁺ ions. 2) Isolated, activated gelsolin domain G3. While not previously reported in the literature, this structure is virtually identical to that observed for activated G3 within the G1-G3:actin complex. 3) Activated recombinant human G4-G6. The source material and crystallization conditions are different from those reported elsewhere, but the results confirm that the C-terminal half of gelsolin can be fully activated in the presence of Ca²⁺, even in the absence of actin. 4) and 5) Two crystal structures of GsnVHH11. This nanobody had not previously been crystallized. The two structures differ in the path of the CDR1 loop that is involved in binding gelsolin. 6) GsnVHH9. The results, from different materials and crystallization conditions than previously reported, confirm the validity of the previous structure. Finally, runs using in silico protein-protein docking software suggest possible binding sites for GsnVHH11 and GsnVHH13 on activated G2-G3 and activated G4-G6, respectively.

Text

2012-08-14

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/42921

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/