The goal of this R21 proposal is to obtain high-quality 1D and 2D crystals of histidine-tagged (His-tag) soluble proteins & His-tag integral membrane proteins (IMP) using a family of new nitrilotriacetic acid (NTA) reagents that promote nucleation in a symmetry-guided manner and accelerate crystallization via phase separation into IMP-rich domains. These studies will serve as a starting point for the long-term goal of generalizing these materials and methods for this challenging class of proteins. One dimensional templates will be used to nucleate the crystallization of soluble proteins such as His-tag green fluorescent protein (His- GFP) in the initial phase of the project. Experience gained with these materials will then be extended to the development of new reagents for the crystallization of the integral membrane protein, human leukotriene C4 synthase, within a two dimensional template matrix. In this case, human leukotriene C4 synthase crystals with improved long-range order will be nucleated in the presence of symmetrical water-soluble nucleating agents that bind a discrete number of proteins in a predetermined geometric arrangement. Once the proteins have been clustered in this symmetry-guided manner, phase-separating lipid mixtures will be used to laterally concentrate the integral membrane protein into protein-rich domains that favor the formation of two dimensional crystals. The materials developed in this proposal will be used to screen for the appearance of crystals with imprinted symmetry and long-range order using cryogenic electron microscopy and x-ray crystallography techniques. Two proteins of known structure, His-GFP at atomic resolution and human leukotriene C4 synthase at medium resolution, will be used as test cases to determine whether the proposed materials promote crystallization of soluble and integral membrane proteins, respectively. The reagents developed in this project will be sent for additional screening with His-tag forms of MalFGK2 maltose transporter, cytochrome b6f, and ribose transporter in our collaborator's laboratories. This 'high risk- high reward' proposal has the potential to revolutionize the field of IMP structural biology, and accelerate the pace of drug development designed to target this important class of proteins, by catalyzing the controlled nucleation and growth of well-ordered His-tag integral membrane proteins of many different types for medium resolution electron crystallography and high resolution x-ray crystallography studies. ? ? ?
|Yu, Guimei; Vago, Frank; Zhang, Dongsheng et al. (2014) Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies. J Struct Biol 187:1-9|
|Grey, Jessica; Thompson, David (2010) Challenges and Opportunities for New Protein Crystallization Strategies in Structure-Based Drug Design. Expert Opin Drug Discov 5:1039-1045|