In collaboration with Dr. Paul Randazzo, we are studying the structure and interactions between Arf proteins and a member of the ASAP family of Arf-GAP proteins. We have produced a variety of stable-isotope variants of these proteins and established conditions for NMR spectroscopic studies. Studies are underway to provide resonance assignments, structure confirmation based on existing known structures and to determine structures which are unknown, binding studies of cofactors, and to examine the assembly of larger multi-domain and multi-component systems. New findings are emerging on the role of the PH domain within ASAP1, which we are pursuing. We are developing methods to examine these proteins in a membrane environment. We will also combine the use of SAXS data with novel NMR and EPR methods to develop three-dimensional structural models of these complexes. We have utilized unique methodology in the NMR spectroscopy, structure calculation and isotopic labeling to obtain new structural information about ASAP1. These data are combined with binding information to activating lipids and membrane mimetics to pursue functional understanding of these important proteins. Recent developments include structural elucidation by X-ray and NMR of the PH domain from ASAP1 interacting with PIP2 lipid head groups. We have successfully demonstrated binding to nano disk membrane mimetics, the equivalence of the mimetic and large unilamellar membrane standards, and we have described a unique cooperative binding mechanism of the ASAP1 PH domain with membrane surfaces. Ongoing work is investigating the assembly of myristoylated-Arf1 into membrane nano disks and the complex of ASAP1 functional domains with this Arf1 at the membrane surface.
| Jian, Xiaoying; Tang, Wai-Kwan; Zhai, Peng et al. (2015) Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1. Structure 23:1977-88 |
| Gruschus, James M; Byrd, R Andrew; Randazzo, Paul A (2014) The importance of seeing surface (effects). Structure 22:363-5 |
