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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Chin, Jean
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Scripps Research Institute
La Jolla
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Shah, Manish B; Zhang, Qinghai; Halpert, James R (2018) Crystal Structure of CYP2B6 in Complex with an Efavirenz Analog. Int J Mol Sci 19:
Ishchenko, Andrii; Peng, Lingling; Zinovev, Egor et al. (2017) Chemically Stable Lipids for Membrane Protein Crystallization. Cryst Growth Des 17:3502-3511
Shah, Manish B; Liu, Jingbao; Zhang, Qinghai et al. (2017) Halogen-? Interactions in the Cytochrome P450 Active Site: Structural Insights into Human CYP2B6 Substrate Selectivity. ACS Chem Biol 12:1204-1210
Maekawa, Keiko; Adachi, Motoyasu; Matsuzawa, Yumiko et al. (2017) Structural Basis of Single-Nucleotide Polymorphisms in Cytochrome P450 2C9. Biochemistry 56:5476-5480
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
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
Bennett, Brad C; Purdy, Michael D; Baker, Kent A et al. (2016) An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels. Nat Commun 7:8770
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
Peng, Lingling; Mo, Fanyang; Zhang, Qinghai (2015) Cholate-based synthesis of size-tunable cage compounds. J Org Chem 80:1221-8
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

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