Albert Einstein College of Medicine

      Mark E. Girvin

      Girvin Laboratory Home Page
      NMR Laboratory Home Page

      Department of Biochemistry

      FORCHHEIMER BLDG.- ROOM 303

      718 430-2025; girvin@aecom.yu.edu

      Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. Atomic-level molecular structures will be required for detailed interpretation of an enormous amount of genetic and chemical information that has been compiled for these proteins. Unfortunately, intrinsic membrane proteins have proven extremely resistant to structural analysis. X-ray studies have been hampered by difficulties in growing crystals of detergent solubilized proteins which are suitable for diffraction. NMR eliminates the need for crystallization, but the size of the protein that can be studied has typically been limited to less than 30 kD. The overall goals of research in this laboratory are to develop and apply new solution conditions and NMR methods to piece together structures of larger membrane proteins and complexes in order to understand how they function.

      Subunit c of the F1F O ATP synthase is one focus of present efforts in the lab, both as a model system and as an interesting protein in its own right. In the ATP synthase, H+ translocation across the membrane through FO provides the driving force for ATP synthesis on F1. Twelve copies of subunit c make up the bulk of the FO complex. This subunit is responsible for both H+ translocation through FO (via ionization of the buried Asp61 residue), and transmission of long range (~100 Å) conformational changes to the ATP binding sites on F1.

      A convenient assay for native folding of membrane proteins in mixed solvents and detergent micelles is being developed, allowing us to optimize for proper folding and for favorable NMR properties before undertaking lengthy structural studies. We have shown that subunit c folds properly in an organic/aqueous solvent mixture, simplifying structural study by NMR. Introduction of a nitroxide at a unique site allowed distances over a 10-20 Å range to be measured, which were used for initial models of the protein's structure. 2D difference NMR methods were applied to another nitroxide-modified form of the protein to rapidly determine the detailed structure of the region of the protein within 16 Å of the probe.

      More recently, we have determined the complete structure of subunit c below the pKa of the essential Asp61 by 1H13 C15N 3D NMR, and are presently solving the structure of the protein above the pKa of Asp61. This pair of structures will provide a detailed picture of the conformational changes linked to energy transduction. The structure of subunit c also provided obvious clues as to how the monomers would pack as an oligomer in the FO complex. A model for the dodecamer was calculated that was consistent with all biochemical for the complex, including extensive cross-linking results.

      The structural features which stabilize the folding of membrane proteins are another interest of the lab. Membrane proteins stand some of the normal folding rules on their heads - e.g. they don't bury hydrophobic surface in their core the way that water soluble proteins do. We expect that clusters of aromatic residues, intricate van der Waals packing, and connecting loops will contribute to folding stability. Each of these elements will be varied by mutagenesis, and folding stability will be assayed by NMR distance and dynamics measurements.

      These methods are being applied to other important membrane proteins including channels, pumps, and photosynthetic light-harvesting complexes.

      Selected Recent Publications (full listing)

      V.K. Rastogi & M.E. Girvin (1999) Structural changes linked to proton translocation by subunit c of the ATP Synthase, Nature 402:263. (Abstract)

      V.K. Rastogi & M.E. Girvin (1999) 1H, 13C, 15N Assignments and Secondary Structure of the High pH Form of Subunit c of the F1FO ATP Synthase, J. Biomolecular NMR 13:91.

      G.P. Wang, S.C. Cahill, X. Liu, M.E. Girvin & C. Grubmeyer (1999) Motional Dynamics of the Catalytic Loop in OMP Synthase, Biochemistry 38:284. (Abstract)

      M.E. Girvin, V.K. Rastogi, F. Abildgaard, J.L. Markley & R.H. Fillingame (1998) Solution Structure of the Transmembrane H+-Translocating Subunit c of the F1FO ATP Synthase, Biochemistry 37:8817. (Abstract)

      G.H. Hockerman, M.E. Girvin, C.C. Malbon, & A.E. Ruoho (1996) Antagonist Conformations within the beta2-Adrenergic Receptor Ligand Binding Pocket, Molecular Pharmacology 49:1021. (Abstract)

      P. Wiedemann, K. Giehl, S.C. Almo, A.A. Federov, M.E. Girvin, P. Shienberger, M. Rudiger, M. Ortner, M. Sippl, & R. Valenta (1996) Molecular and Structural Analysis of a Continuous Birch Pollen Profilin Epitope Defined by a Monoclonal Antibody, J. Biol. Chem. 271: 29915. (Abstract)

      M.E. Girvin & R.H. Fillingame (1995) Determination of Local Protein Structure by Spin Label Difference 2D NMR, Biochemistry 34:1635. (Abstract)

      M.E. Girvin (1994) Increased Sensitivity of COSY Spectra by Use of Constant Time t1 Periods (CT-COSY), J. Magnetic Resonance Ser. A. 108:99.

      File Updated 09/99