Membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport - making them important players in infectious disease, genetic disorders, and cancer. Despite their importance, their numbers (20 to 35% of the protein sequences in all genomes), and the need for structure to understand their function, they make up only 0.2% of the structures in the Protein Data Bank. Obviously these proteins have resisted routine structural analysis. The overall research goals of this laboratory are to develop and apply new solution conditions and NMR methods to determine structures of larger membrane proteins and complexes, particularly transporters and pumps, in order to understand how they function.

The FO portion of the F1FO ATP synthase is one focus of present efforts in the lab, both as a model system and because of its importance. This complex is responsible for synthesizing the vast majority of cellular ATP - over 80 pounds of ATP per day in the average human. In the ATP synthase, H+ translocation across the membrane through FO provides the driving force for ATP synthesis on F1. A ring of ten 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 generation of the long range conformational changes transmitted over 100 Å to the ATP binding sites on F1. We have determined the complete structures of subunit-c below and above the pKa of Asp61, providing a detailed picture of the conformational changes linked to energy transduction. The c-subunit of the ATP synthase was recently shown to be a useful, genus-specific target for antibiotics (against M. tuberculosis, for example), and so we are also investigating the structural basis for the specificity of inhibitors with the human, M. tuberculosis, and E. coli homologs of the protein.

A second transporter that we are studying in the lab is a relatively simple but biomedically important small multidrug resistance pump from Staphylococcus aureus. Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug Resistant Pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug Resistance pumps (SMRs) are 100-110 residue dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. We are determining the structures of the conformations that make up the functional cycle of an SMR, and identifying the binding determinants for multiple drugs and inhibitors using solution NMR.

S. aureus Small MDR

Poget, S.F., Cahill, S.M., and Girvin, M.E. (2007) Isotropic bicelles stabilize the functional form of a small multidrug-resistance pump for NMR structural studies. J Am Chem Soc. 129, 2432-2433.

Kamen, D.E., Cahill, S.M., and Girvin, M.E. (2007) Multiple alignment of membrane proteins for measuring residual dipolar couplings using lanthanide ions bound to a small metal chelator. J Am Chem Soc. 129, 1846-1847.

Poget, S.F., Krueger-Koplin, S.T., Krueger-Koplin, R.D., Cahill, S.M., Shekar, S.C., and Girvin, M.E. (2006) NMR Assignment of the Dimeric S. aureus Small Multidrug-Resistance Pump in LPPG Micelles. J Biomol NMR. 2006 Feb 2.

Shekar, S.C., Wu, H., Fu, Z., Yip, S.C., Nagajyothi, Cahill, S.M., Girvin, M.E., Backer, J.M. (2005) Mechanism of constitutive phosphoinositide 3-kinase activation by oncogenic mutants of the p85 regulatory subunit. J. Biol. Chem. 280, 27850-27855.

Kamen, D.E., Gross, S.T., Girvin, M.E. and Wilson, D.W. (2005) Structural basis for the physiological temperature-dependence of VP16 association with the cytoplasmic tail of Herpes simplex virus glycoprotein H, J. Virol. 79, 6134-6141.

Rivera-Torres, I.O., Krueger-Koplin, R.D., Hicks, D.B., Cahill, S.M., Krulwich, T.A., and Girvin, M.E. (2004) pKa of the Essential Glu54 and Backbone Conformation for Subunit c from the H+-coupled F1Fo ATP Synthase from an Alkaliphilic Bacillus, FEBS Lett, 575, 131-135.

Krueger-Koplin, R.D., Sorgen, P.L., Cahill, S.M., and Girvin, M.E. (2004) An evaluation of detergents for NMR structural studies of membrane proteins J. Biomol. NMR, 28, 43-57.

Deng H, Lewandowicz A, Cahill SM, Furneaux RH, Tyler PC, Girvin ME, Callender RH, Schramm VL. (2004) Assignment of Downfield Proton Resonances in Purine Nucleoside Phosphorylase-Immucillin-H Complex by Saturation-Transferred NOEs, Biochemistry 43, 1980-1987.

Sauve, A.A., Cahill, S.M., Basso, L.A., Santos, D.S., Grubmeyer, C., Evans, G.B., Furneaux, R.H., Tyler, P.C., Girvin, M.E., and Schramm, V.L. (2003) Ionic States of Substrates and Transition State Analogues at the Catalytic Sites of N-Ribosyltransferases Biochemistry 42, 5694-5705.

Sorgen, P.L., Hu, Y., Guan, L., Kaback, H.R., and Girvin, M.E. (2002) An approach to membrane protein structure without crystals Proc. Natl. Acad. Sci. USA 99, 14037-14040.

Duffy HS, Sorgen PL, Girvin ME, O'Donnell P, Coombs W, Taffet SM, Delmar M, Spray DC. (2002) pH-dependent intramolecular binding and structure involving Cx43 cytoplasmic domains J. Bio. Chem. 277, 36706-36714 (Abstract)

Sorgen PL, Duffy HS, Cahill, SM, Coombs W, Spray DC, Delmar M, Girvin ME Sequence specific resonance assignment of the carboxyl terminal domain of Connexin43. (2002) J. Biomol. NMR. 23:245.

Sorgen, P.L., Cahill, S.M., Krueger-Koplin, R.D., Krueger-Koplin, S.T., Schenck, C.S., & Girvin, M.E. (2002) Structure of the Rhodobacter sphaeroides Light Harvesting-1 beta subunit in detergent micelles Biochemistry 41:31.

Schon, E.A., Santra, S., Pallotti, F., & Girvin, M.E. (2001) Pathogenesis of primary defects in mitochondrial ATP synthesis Cell & Devel. Biol. 12:441. (Abstract)

Girvin, M.E. (2001) ATP Synthase, in The Encyclopedia of Molecular Medicine, T.E. Creighton Ed. J. Wiley & Sons, pp. 287-290.

Taylor, R.M., Zakharov , S.D., Heymann, J.B., Girvin, M.E., & Cramer, W.A. (2000) Folded State of the Integral Membrane Colicin E1 Immunity Protein in Solvents of Mixed Polarity, Biochemistry, 39:12131. (Abstract)

Mahoney, N.M., Rastogi, V.K., Cahill, S.M., Girvin, M.E. & Almo, S.C. (2000) A New Method to Determine the Binding Orientation of Proline-Rich Ligands in Solution: Polarity of the Profilin-Ligand Interaction, J. Am. Chem. Soc. 122:7851.

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 F₁F_O 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)

Barlow, J.N., Girvin, M.E., & Blanchard, J.S. (1999) Positional isotope exchange catalyzed by UDP-galactopyranose mutase, J. Am. Chem. Soc. 121, 6968.

Shi, W., Li, C.M., Tyler, P.C., Furneaux, R.H., Cahill, M.C., Girvin, M.E., Grubmeyer, C., Schramm, V.L., & Almo, S.C. (1999) The 2.0 Å Structure of Malarial Purine Phosphoribosyltransferase in Complex with a Transition-State Analogue Inhibitor, Biochemistry 38, 9872. (Abstract)

Li, C.M., Tyler, P.C., Furneaux, R.H., Kicska, G., Xu, Y., Grubmeyer, C., Girvin, M.E. & Schramm, V.L. (1999) Transition-state analogues as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases, Nat. Struct. Biol. 6, 582. (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 F₁F_O ATP Synthase, Biochemistry 37:8817. (Abstract)