Thanks to NIH funding support
"Mother Nature's Blowtorch"
Membrane proteins present one of the principal challenges for structural biology today. Conventional methods of structure elucidation, i.e. X-ray crystallography and solution-state NMR, usually fail when applied to membrane proteins. Solid-state NMR spectroscopy is rapidly evolving as the method of choice for structure determinations of membrane proteins. The Ramamoorthy lab has played a central role in the development of solid-state NMR and is currently applying solid-state NMR to the structure elucidation of membrane proteins.
"Mother Nature's Blowtorch"
Catabolism of numerous endo- and exogenous substances, including many drugs and toxins, is catalyzed by the cytochrome P450 family of enzymes. The scheme below shows the oxidation of cholesterol into a more soluble derivative; this is the first step in the degradation of cholesterol to bile acids.
Also shown in the scheme are the electron carrier proteins cytochrome b5 and Cytochrome P450 reductase which supply the two electrons needed by cytochrome P450 to perform its function.
Also shown in the scheme are the electron carrier proteins cytochrome b5 and Cytochrome P450 reductase which supply the two electrons needed by cytochrome P450 to perform its function.
Structural Biology of Full-length Cytochrome b5 and Cytochrome P450
Cytochrome b5 is an important electron carrier membrane associated protein whose intimate interactions with cytochrome P450 facilitate various catalytic oxidative reactions in the microsomes. This 16.7 kDa protein consists of three distinct regions: a water-soluble heme domain, a 14-residue linker and a transmembrane alpha-helix. The water-soluble domain of cytochrome b5 displays significant homology to both hemoglobin and myoglobin and can be easily cleaved off via trypsin. In its native form, the water-soluble domain of cytochrome b5 is anchored to the endoplasmic reticulum membrane via a hydrophobic transmembrane helix. This helix seems to play a critical role in the proper functioning of cytochrome b5 as evident in previous studies.
Some of the goals of this project include providing:
1) The high-resolution structures of Cytochrome b5 and Cytochrome P450 in magnetically aligned bicelles.
2) The high-resolution structure of Cytochrome b5:Cytochrome P450 complex in magnetically aligned bicelles.
3) Information on the dynamics of the Cytochrome b5 and Cytochrome P450, individually, in lipid bilayers.
4) Information on the dynamics and interactions of the Cytochrome b5:Cytochrome P450 complex in lipid bilayers.
5) Information on the dynamics and interactions of the Cytochrome P450 : Cytochrome P450 Reductase complex in lipid bilayers.
2) The high-resolution structure of Cytochrome b5:Cytochrome P450 complex in magnetically aligned bicelles.
3) Information on the dynamics of the Cytochrome b5 and Cytochrome P450, individually, in lipid bilayers.
4) Information on the dynamics and interactions of the Cytochrome b5:Cytochrome P450 complex in lipid bilayers.
5) Information on the dynamics and interactions of the Cytochrome P450 : Cytochrome P450 Reductase complex in lipid bilayers.
Publications on this project
- Dürr UHN, Soong R, Ramamoorthy A. When the detergent meets bilayer: Birth and coming of age of lipid bicelles. Prog. NMR Spec. 69(2013) 1-22.
- Pandey M, Ramamoorthy A. Quantum chemical calculation of amide-15N chemical shift anisotropy tensors for a membrane-bound cytochrome b5. J. Phys. Chem. B. 117(2013) 859-867.
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Dürr UHN, Gildenberg M, Ramamoorthy A. The magic of bicelles lights up membrane protein structure. Chem. Rev. 112 (2012) 6054-6074.
- Pandey M, Vivekanandan S, Ahuja S, Kumar P, Waskell L, Im S-C, Ramamoorthy A. Determination of 15NChemical Shift Anisotropy from a Membrane Bound Protein by NMR Spectroscopy. J. Phys. Chem. 116(2012) 7181-7189.
- Veglia G and Ramamoorthy A. Preface: Special issue on Membrane Protein Dynamics: Correlating Structure to Function Biochim. Biophys. Acta 2 (2010) 65-67.
- 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, 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.
- 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.
Publications on other membrane proteins the Ramamoorthy Lab has worked on in the past
- Kandasamy SK, Lee D-K, Nanga RPR, Xu J, Santos JS, Larson R, Ramamoorthy A. Solid-state NMR and Molecular Dynamics Simulations Reveal the Oligomeric Ion-channels of TM2-GABAA Stabilized by Intermolecular Hydrogen Bonding. Biochim. Biophys. Acta. 1788 (2009) 686-695.
- Ramamoorthy A, Kandasamy SK, Lee DK, Kidambi S, Larson RG, Structure,Topology and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies, Biochemistry 46 (2007) 965-975.
- Pointer-Keenan CD, Lee DK, Hallock K, Tan AM, Zand R, Ramamoorthy A, Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy, Chem. Phys. Lip. 132 (2004) 47-54.