Membrane proteins are essential regulators of a number of cellular and physiological processes including signaling between cells, transport across cell membranes and energy transduction processes. High-resolution structures of membrane proteins can provide insight into their function and enable the development of compounds to alter their properties for biological and biomedical purposes. While more than 30% of the human genome and 50% of known drug targets are membrane proteins, few structures of membrane proteins are known. For most membrane proteins, this is due to the lack of membrane mimetics that contain the protein in a stable, functional, and homogeneous state. In this application, we propose to develop robust approaches to produce near-native model membranes that both stabilize membrane-bound proteins or protein-protein complexes in their functional forms and will also enable structure determination at high-resolution by NMR spectroscopy. We have chosen cytochrome proteins (CytP450, CytP450-reductase (CYPOR) and Cytb5) and their complexes (P450-CYPOR and P450- b5) as model systems for optimization. Challenges posed by these membrane proteins are manifold and therefore the biochemical and biophysical approaches developed to tackle these challenges can be relatively easily extended for structural studies of other membrane proteins. To accomplish this goal, we propose to investigate new classes of mild detergents and non-detergent surfactants as well as native membrane-like bicelles that show promise in stabilizing the native fold of membrane proteins using a rapid assignment-free screening process by NMR spectroscopy. While the focus of this proposal is on the rapid optimization of sample conditions for NMR structural studies, the outcome can be extended for investigations using a variety of other biophysical techniques including EPR, FRET and X-ray crystallography.

Public Health Relevance

The outcome of the proposed studies on the production and optimization of membrane proteins will significantly advance our ability to obtain structural and functional insights at atomic-level resolution and will aid in the development of drugs to treat various diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095640-04
Application #
8520339
Study Section
Special Emphasis Panel (ZRG1-BCMB-S (50))
Program Officer
Preusch, Peter C
Project Start
2010-09-30
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$288,090
Indirect Cost
$97,986
Name
University of Michigan Ann Arbor
Department
Biophysics
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Yamamoto, Kazutoshi; Caporini, Marc A; Im, Sang-Choul et al. (2015) Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochim Biophys Acta 1848:342-9
Zhang, Rongchun; Damron, Joshua; Vosegaard, Thomas et al. (2015) A cross-polarization based rotating-frame separated-local-field NMR experiment under ultrafast MAS conditions. J Magn Reson 250:37-44
Huang, Rui; Yamamoto, Kazutoshi; Zhang, Meng et al. (2014) Probing the transmembrane structure and dynamics of microsomal NADPH-cytochrome P450 oxidoreductase by solid-state NMR. Biophys J 106:2126-33
Higashi, Kenjirou; Yamamoto, Kazutoshi; Pandey, Manoj Kumar et al. (2014) Insights into atomic-level interaction between mefenamic acid and eudragit EPO in a supersaturated solution by high-resolution magic-angle spinning NMR spectroscopy. Mol Pharm 11:351-7
Lee, Sanghyun; Zheng, Xueyun; Krishnamoorthy, Janarthanan et al. (2014) Rational design of a structural framework with potential use to develop chemical reagents that target and modulate multiple facets of Alzheimer's disease. J Am Chem Soc 136:299-310
Savelieff, Masha G; Liu, Yuzhong; Senthamarai, Russell R P et al. (2014) A small molecule that displays marked reactivity toward copper- versus zinc-amyloid-* implicated in Alzheimer's disease. Chem Commun (Camb) 50:5301-3
Zhang, Rongchun; Ramamoorthy, Ayyalusamy (2014) Performance of RINEPT is amplified by dipolar couplings under ultrafast MAS conditions. J Magn Reson 243:85-92
Pandey, Manoj Kumar; Vivekanandan, Subramanian; Yamamoto, Kazutoshi et al. (2014) Proton-detected 2D radio frequency driven recoupling solid-state NMR studies on micelle-associated cytochrome-b(5). J Magn Reson 242:169-79
Zhao, Jun; Hu, Rundong; Sciacca, Michele F M et al. (2014) Non-selective ion channel activity of polymorphic human islet amyloid polypeptide (amylin) double channels. Phys Chem Chem Phys 16:2368-77
Nishiyama, Yusuke; Malon, Michal; Ishii, Yuji et al. (2014) 3D ¹?N/¹?N/¹H chemical shift correlation experiment utilizing an RFDR-based ¹H/¹H mixing period at 100 kHz MAS. J Magn Reson 244:1-5

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