NMR Methodology

Thanks to NIH funding support

Cyt-b5 is a 16.7kDa membrane-bound protein which participates in and modifies the catalytic activity of cytochrome P450 (Cyt-P450); Cyt-P450 enzymes metabolize about half of the drugs in clinical use today. Cyt-b5 is also involved as an electron transfer component in a number of oxidative reactions in biological tissues which includes the biosynthesis of fatty acids and steroids. Cyt-b5 consists of a catalytic heme-containing domain, a membrane-anchor domain and a linker region which connects the two. Solubilization of Cyt-b5 by removal of the membrane anchor, through protease cleavage of the linker region, generates a protein that is incapable of electron transfer to microsomal Cyt-P450. This dramatic change in the properties of Cyt-b5 upon elimination of its transmembrane domain indicates that it plays an essential role in the function of Cyt-b5. In order to better understand the molecular mechanism by which Cyt-b5 influences the catalysis of Cyt-P450, we propose to solve the structure of the membrane-associated rabbit Cyt-b5 in the absence and determine the structural changes in the presence of rabbit Cyt-P450 2B4 (molecular weight is 55.755 kDa) using NMR experiments.

Our lab has been developing NMR methods for the structural studies of membrane proteins, and has the expertise to study the structure of Cyt-b5. Protein production and functional measurements are being carried out in collaboration with Dr. Waskell at the University of Michigan. We are presently investigating:

three-dimensional structure of Cyt-b5
structure and orientation of Cyt-b5in lipid bilayers.
dynamics of membrane-bound Cyt-b5.
structure of membrane-bound Cyt-b5 in a complex with Cyt-P450.

These studies will lead to a detailed understanding of how Cyt-b5, a membrane protein with a single transmembrane domain, interacts with the membrane and influences the activity of its cytosolic domain. Furthermore, these experiments will shed light on how Cyt-b5 participates in the mechanism of the electron transfer reaction that underlies catalysis by Cyt-P450. The outcome should also clarify how the sequence of the membrane anchor restricts its cellular location. The challenges posed by these systems would enable us to develop novel NMR methods that can be used for studying significant other membrane proteins as well.

“An innovative NMR spectroscopy method to speed up data acquisition from membrane proteins”

This (Ritchey award winning) cutting-edge approach utilizes unpaired electrons to light up nuclear spins of membrane-associated proteins, and enable easy detection of atomic-level resolution structures.

Publications from this project

Nishiyama Y, Zhang R, Ramamoorthy A. Finite-pulse radio frequency driven recoupling with phase cycling for 2D 1H/1H correlation at ultrafast MAS frequencies. J. Magn. Reson. (In press)
Yamamoto K, Paige P, Ramamoorthy A. Bicelles exhibiting magnetically-alignment for a broader range of temperatures - A solid state NMR study. Langmuir. (In press).
Yamamoto K, Vivekanandan S, Ramamoorthy A. Fast NMR Data Acquisition From Bicelles Containing a Membrane-Associated Peptide at Natural-Abundance. J. Phys. Chem. B. 115 (2011) 12448-12455.
Khitrin A, Xu J, Ramamoorthy A. Cross-Correlations Between Low-gamma Nuclei in Solids Via a Common Dipolar Bath. J. Magn. Reson. 212 (2011) 95-101.
Xu J, Smith PES, Soong R, Ramamoorthy A. A Proton Diffusion Based Solid-State NMR Approach for Structural Studies on Aligned Samples. J. Phys. Chem. B. 115 (2011) 4863-4871.
Xu J, Soong R, Im SC, Waskell L, Ramamoorthy A. INEPT-Based Separated-Local-Field NMR Spectroscopy: A Unique Approach to Elucidate Side-Chain Dynamics of Membrane-Associated Proteins. J. Am. Chem. Soc. 132 (2010) 9944-9947.
Saito H, Ando I, Ramamoorthy A. Chemical shift tensor - The heart of NMR: Insights into biological aspects of proteins. Prog. NMR Spec. 57 (2010) 181-228.
Yamamoto K, Xu J, Kawulka KE, Vederas JC, Ramamoorthy A. Use of a copper-chelated lipid speeds up NMR measurements from membrane proteins. J. Am. Chem. Soc. 132 (2010) 6929-6931.
Soong R, Smith PE, Xu J, Yamamoto K, Im SC, Waskell L, Ramamoorthy A. Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information. J. Am. Chem. Soc. 132 (2010) 5779-5788.
Fukami T, Ishii T, Io T, Suzuki N, Suzuki T, Yamamoto K, Xu JD, Ramamoorthy A, Tomono K. Nanoparticle Processing in the Solid State Dramatically Increases the Cell Membrane Permeation of a Cholesterol-Lowering Drug. Probucol. Mol. Pharmaceutics 6 (2009) 1029-1035.
Xu J, Dürr UHN, Im SC, Gan Z, Waskell L, Ramamoorthy A. Bicelle-Enabled Structural Studies on Membrane Protein Cytochrome b5 by Solid-State MAS NMR Spectroscopy. Angewandte Chemie-International Edition. 47 (2008) 7864-7867.
Dürr UHN, Yamamoto K, Im SC, Waskell L, Ramamoorthy A, Solid-State NMR Reveals Structural and Dynamical Properties of a Membrane-Anchored Electron-Carrier Protein, Cytochrome b(5). J. Am. Chem. Soc. 129 (2007) 6670-6671.
Dürr UHN, Waskell L, Ramamoorthy A. The cytochromes P450 and b5 and their reductases-Promising targets for structural studies by advanced solid-state NMR spectroscopy. Biochim. Biophys. Acta. 1768 (2007) 3235-3259.
Xu J, Struppe J, Ramamoorthy A. Two-dimensional homonuclear chemical shift correlation established by the cross-relaxation driven spin diffusion in solids. J. Chem. Phys. 128 (2008) 052308.