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
Zhang, Rongchun; Mroue, Kamal H; Ramamoorthy, Ayyalusamy (2016) Hybridizing cross-polarization with NOE or refocused-INEPT enhances the sensitivity of MAS NMR spectroscopy. J Magn Reson 266:59-66
Pandey, Manoj Kumar; Yarava, Jayasubba Reddy; Zhang, Rongchun et al. (2016) Proton-detected 3D (15)N/(1)H/(1)H isotropic/anisotropic/isotropic chemical shift correlation solid-state NMR at 70kHz MAS. Solid State Nucl Magn Reson 76-77:1-6
Zhang, Rongchun; Ramamoorthy, Ayyalusamy (2016) Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS. J Chem Phys 144:034202
Zhang, Meng; Huang, Rui; Ackermann, Rose et al. (2016) Reconstitution of the Cytb5-CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy. Angew Chem Int Ed Engl 55:4497-9
Korshavn, Kyle J; Bhunia, Anirban; Lim, Mi Hee et al. (2016) Amyloid-β adopts a conserved, partially folded structure upon binding to zwitterionic lipid bilayers prior to amyloid formation. Chem Commun (Camb) 52:882-5
Zhang, Rongchun; Mroue, Kamal H; Ramamoorthy, Ayyalusamy (2015) Proton chemical shift tensors determined by 3D ultrafast MAS double-quantum NMR spectroscopy. J Chem Phys 143:144201
Zhang, Meng; Huang, Rui; Im, Sang-Choul et al. (2015) Effects of membrane mimetics on cytochrome P450-cytochrome b5 interactions characterized by NMR spectroscopy. J Biol Chem 290:12705-18
Zhang, Rongchun; Nishiyama, Yusuke; Sun, Pingchuan et al. (2015) Phase cycling schemes for finite-pulse-RFDR MAS solid state NMR experiments. J Magn Reson 252:55-66
Zhang, Rongchun; Ramamoorthy, Ayyalusamy (2015) Selective excitation enables assignment of proton resonances and (1)H-(1)H distance measurement in ultrafast magic angle spinning solid state NMR spectroscopy. J Chem Phys 143:034201
Huang, Rui; Zhang, Meng; Rwere, Freeborn et al. (2015) Kinetic and structural characterization of the interaction between the FMN binding domain of cytochrome P450 reductase and cytochrome c. J Biol Chem 290:4843-55

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