Compared to soluble proteins, it is much more challenging to obtain well diffracting membrane protein crystals suitable for X-ray analysis. Membrane protein crystals are characterized by small hydrophilic protein- protein interactions that are crucial for formation of a 3D crystal lattice. Approaches to expand or modify the polar surface of membrane proteins are effective for crystal growth but still represent significant challenges. The overarching goal of our proposal is to develop an innovative approach of using intelligently designed amphiphiles or lipids, the essential component required to stabilize membrane proteins, to mediate ordered protein surface interactions so as to increase the crystallization propensity and improve crystal diffraction. This approach is orthogonal and complementary to available protein engineering techniques and applicable to both detergent micelle and lipid bilayer based crystallization protocols. To achieve our goal, we will develop new design principles for the creation of novel amphiphiles. We will use biochemical assays and various biophysical techniques to study the thermodynamic interaction and binding between the amphiphiles and membrane proteins, as these properties govern protein stability and function. Crystallization experiments will be performed to identify molecules that mediate ordered membrane protein crystal contacts and reveal molecular details of the amphiphile-protein interaction. Through this work, structurally novel stabilization reagents will be developed to overcome the crystallization bottleneck that cannot be fully addressed by currently available detergents, lipids or other novel amphiphiles that have been tested in the last two decades.
We aim to identify a robust set of reagents that can be generally applicable to the structural solution of different families of membrane proteins, not ones limited to a single protein or a single class. By improving the resolution of previously solved structures and facilitating the structural determination of new membrane proteins, our study will have a direct impact on biology.
Membrane proteins account for about one third of human genes and comprise more than 50% of human drug targets. High-resolution structures of membrane proteins are required to understand the underlying biological and molecular mechanisms and facilitate structure-based drug design efforts. Structural information is currently quite difficult to acquire, therefore the development of new methodologies and reagents in this area is critical to facilitate this process.
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|Shah, Manish B; Jang, Hyun-Hee; Wilderman, P Ross et al. (2016) Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry. Biophys Chem 216:1-8|
|Shah, Manish B; Liu, Jingbao; Huo, Lu et al. (2016) Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism. Mol Pharmacol 89:435-45|
|Liu, Jingbao; Shah, Manish B; Zhang, Qinghai et al. (2016) Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations. Biochemistry 55:1997-2007|
|Peng, Lingling; Mo, Fanyang; Zhang, Qinghai (2015) Cholate-based synthesis of size-tunable cage compounds. J Org Chem 80:1221-8|
|Moeller, Arne; Lee, Sung Chang; Tao, Houchao et al. (2015) Distinct conformational spectrum of homologous multidrug ABC transporters. Structure 23:450-60|
|Shah, Manish B; Wilderman, P Ross; Liu, Jingbao et al. (2015) Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison. Mol Pharmacol 87:649-59|
|Szewczyk, Paul; Tao, Houchao; McGrath, Aaron P et al. (2015) Snapshots of ligand entry, malleable binding and induced helical movement in P-glycoprotein. Acta Crystallogr D Biol Crystallogr 71:732-41|
|Wang, An; Stout, C David; Zhang, Qinghai et al. (2015) Contributions of ionic interactions and protein dynamics to cytochrome P450 2D6 (CYP2D6) substrate and inhibitor binding. J Biol Chem 290:5092-104|
|Hildebrandt, Ellen; Zhang, Qinghai; Cant, Natasha et al. (2014) A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR). Biochim Biophys Acta 1838:2825-37|
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