Bacteriorhodopsin, spectroscopy, protein crystallography
Dr. Lanyi studies the physiology, biochemistry, and the molecular mechanisms of ion translocation across biological membranes. His work focuses on the light driven electrogenic ion pump in the cytoplasmic membrane of Halobacterium halobium, bacteriorhodopsin, an outward directed proton pump). This purple pigment is a small protein in which retinal isomerization after photon absorption initiates molecular rearrangements and results in ion uptake on one side of the membrane, translocation, then release on the other side.
Individual steps of the photocycles are followed by flash-spectroscopy with a gated optical multichannel analyser, which reveals the rise and decay of several intermediate species with red- and blue-shifted absorption spectra. The measured difference spectra are deconvoluted, using computer intensive grid-search, into component spectra. The relative weights of these spectra are the time-dependent concentrations of the intermediates, from which the kinetics are reconstructed. The kinetic models reveal the steps for internal transfers of proton and chloride, and their release and uptake at the membrane surfaces during transport. The temperature dependencies of the rate constants provide information on the thermodynamics of energy coupling between the photoexcited retinal chromophore and the proton gradient created.
The kinetic and thermodynamic models for ion translocation, and the spectroscopic measurements, provide the framework for Dr. Lanyi's studies on the mechanistic aspects of the transport. The roles of individual amino acid residues are explored with site-specific mutagenesis, using a newly developed shuttle vector based on a halobacterial plasmid. Altered genes are expressed in H. halobium, which produces the proteins in large quantities and in a wild-type-like two-dimensional membrane array suitable also for diffraction studies of structure. Recent work has concentrated on growing 3-dimensional bacteriorhodopsin crystals. From these, high-resolution x-ray diffraction structures have been determined for seven of the nine states of the photocycle. Together with lower resolution or indirect structural models for the reminaing two states, these add up to an atomic level "movie," that describes the transport mechanism in detail. The results indicate that after absorption of light the all-trans retinal in bacteriorhodopsin is isomerized to 13-cis. This would strongly change the shape of the retinal chain, but the constraints of the binding site do not allow it. The retinal is twisted instead, retaining energy from the absorbed photon. During the first half of the reaction cycle, the binding site gives way and the retinal can relax. In this process, energy is transferred to the protein and that is used for the transport. The electrostatic conflict with the binding site is resolved by transfer of the retinal Schiff base proton to the counter-ion asp-85, allowing the rotation of the unprotonated Schiff base away from asp-85 and toward the other side of the membrane. Protonation of asp-85 causes flipping of the positively charged arg-82 toward a network of water molecules, from which a proton is released to the extracellular membranbe surface. The steric conflict with the binding site is resolved by movement of residues, like trp-182, that allow development of the bent shape of the 13-cis retinal. The repacking of the side-chains causes cavities to arise in the cytoplasmic region, and they become great enough to accumulate water molecules. The resulting single-file chain of water conducts a proton from asp-96 near the cytoplasmic surface to reprotonate the Schiff base. The remainder of the cycle then resets the pump to its initial state.
V. Krishnamani and J. K. Lanyi. Structural Changes in Bacteriorhodopsin during its In Vitro Refolding from a Partially Denatured State. Biophys. J. 100: 1559-1567, 2011.
E. S. Imasheva, S. P. Balashov, J. M. Wang and J. K. Lanyi. Removal and Re-constitution of the Carotenoid Antenna of Xanthorhodopsin. J.Membr. Biol. 239: 95-104, 2011.
S. P. Balashov, E. S. Imasheva, A. R. Choi, K.-H. Jung, S. Liaaen-Jensen and J. K. Lanyi. Reconstitution of Gloeobacter Rhodopsin with Echinenone: Role of the 4-keto Group. Biochemistry 49: 9792-9799, 2010.
A. K. Dioumaev, J. M. Wang and J. K. Lanyi. Low-temperature FTIR Study of Multiple K Intermediates in the Photocycles of Bacteriorhodopsin and Xan-thorhodopsin. J. Phys. Chem. B. 114:2920-2931, 2010.
A. K. Dioumaev and J. K. Lanyi. Two Bathointermediates of the Bacteriorhodopsin Photocycle, from Time-resolved Nanosecond Spectra in the Visible. J. Phys. Chem. B. 113:16643-16653, 2009.
E. S. Imasheva, S. P. Balashov, A. R. Choi, K.-H. Jung and J. K. Lanyi. Reconstitution of Gloeobacter violaceus Rhodopsin with a Light-harvesting Antenna. Biochemistry 48:10948-10955, 2009.
D. Chen and J. K. Lanyi. Structural Changes in the N and N’ States of the Bacteriorhodopsin Photocycle. Biophys. J. 96: 2779-2788, 2009.
T. Polívka, S. P. Balashov, P. Chábera, E. S. Imasheva, A. Yartsev, V. Sundström and J. K. Lanyi. Femtosecond Carotenoid to Retinal Energy Transfer in Xanthorhodopsin. Biophys.J. 96: 2268-2277, 2009.
J. K. Lanyi. What is the Real Crystallographic Structure of the L Photointermediate of Bacteriorhodopsin? Biochim. Biophys. Acta 1658: 14-22, 2004.
S. P. Balashov, E. S. Imasheva, V. A. Boichenko, J. Antón, J. M. Wang and J. K. Lanyi. Xanthorhodopsin: A Proton Pump with a Light-harvesting Carotenoid Antenna. Science 309: 2061-2064, 2005.
S. P. Balashov, E. S. Imasheva, and J. K. Lanyi. Induced Chirality of the Light-harvesting Carotenoid Salinixanthin and its Interaction with the Retinal of Xanthorhodopsin. Biochemistry. 45: 10998-1004, 2006.
J. K. Lanyi and B. Schobert B. Propagating Structural Perturbation Inside Bacteriorhodopsin: Crystal Structures of the M State and the D96A and T46V Mutants. Biochemistry. 45: 12003-12010, 2006.
J. K. Lanyi and B. Schobert. Structural Changes in the L Photointermediate of Bacteriorhodopsin. J. Mol. Biol. 365: 1379-1392, 2007.
D. Chen, J. M. Wang and J. K. Lanyi. Electron Paramagnetic Resonance Study of Structural Changes in the O Photointermediate of Bacteriorhodopsin J. Mol. Biol. 366: 790-805, 2007.
A. K. Dioumaev and J. K. Lanyi. Bacteriorhodopsin Photocycle at Cryogenic Temperatures Reveals Distributed Barriers of Conformational Substates. Proc. Natl. Acad. Sci. U.S.A. 104: 9621-9626, 2007.
H. Luecke, B. Schobert, J. Stagno, E. S. Imasheva, J. M. Wang, S. P. Balashov and J. K. Lanyi Crystallographic Structure of Xanthorhodopsin, the Light-Driven Proton Pump with a Dual Chromophore. Proc. Natl. Acad. Sci. U. S. A. 105: 16561-16565, 2008.
J. K. Lanyi and B. Schobert. Local-global Conformational Coupling in a Heptahelical Membrane Protein: Transport Mechanism from the Crystal Struc-tures of the Nine States of the Bacteriorhodopsin Photocycle. Biochemistry 43: 3-8, 2004.
S. P. Balashov, E. S. Imasheva, J. M. Wang, and J. K. Lanyi. Excited-state Energy Transfer and the Relative Orientation of Retinal Carotenoid in Xanthorhodopsin. Biophys.J. 95: 2402-2414, 2008.
B. Schobert, L.S. Brown and J.K. Lanyi. Crystallographic Structures of the M and N Intermediates of Bacteriorhodopsin: Assembly of a Hydrogen-bonded Chain of Water Molecules Between Asp-96 and the Retinal Schiff base. J. Mol. Biol. 330: 553-570, 2003.
J. K. Lanyi and B. Schobert. Transport Mechanism of Bacteriorhodopsin from Crystal Structures of the K, L, M1, M2 and M2’ Intermediates of the Photocycle. J. Mol. Biol. 328: 439-450, 2003.
A. K. Dioumaev and J. K. Lanyi. Switch from Conventional to Distributed Kinetics in the Bacteriorhodopsin Photocycle. Biochemistry 47:11125-11133, 2008.
L. S. Brown, R. Needleman and J. K. Lanyi. Conformational Change of the E-F Interhelical Loop in the M Photointermediate of Bacteriorhodopsin. J. Mol. Biol. 317: 471-478, 2002.
E.S. Imasheva, S. P. Balashov, J.M. Wang, E. Smolensky, M. Sheves and J. K. Lanyi. Chromophore Interaction in Xanthorhodopsin-Retinal Dependence of Salinixanthin Binding. Photochem Photobiol. 84: 977-984, 2008.
J. K. Lanyi and B. Schobert. Crystallographic Structure of the Retinal and the Protein After Deprotonation of the Schiff Base: the Switch in the Bacteri-orhodopsin Photocycle. J. Mol. Biol. 321, 727-737, 2002.
S. P. Balashov, E. S. Imasheva, J. M. Wang, and J. K. Lanyi. Carotenoid to Retinal Excited-state Energy Transfer in Xanthorhodopsin. Biophys.J. 95: 2402-2414, 2008.
B. Schobert, J. Cupp-Vickery, V. Hornak, S. O. Smith and J. K. Lanyi. Crystallographic Structure of the K Intermediate of Bacteriorhodopsin: Conservation of Free Energy after Photoisomerization of the Retinal. J. Mol. Biol. 321: 715-726, 2002.
J. K. Lanyi. Molecular Mechanism of Ion Transport in Bacteriorhodopsin: Insights from Crystallographic, Spectroscopic and Mutational Studies. J. Physical Chemistry B 104: 11441-11448, 2000.
H. Luecke, B. Schobert, J.-P. Cartailler, H.-T. Richter, A. Rosengarth, R. Needleman and J.K. Lanyi. Coupling Photoisomerization of Retinal to Directional Transport in Bacteriorhodopsin. J.Mol.Biol. 300: 1237-1255, 2000.
H. Luecke, B. Schobert, H. -T. Richter, J. -P. Cartailler and J. K. Lanyi. Structural Changes in the M Photointermediate of Bacteriorhodopsin at 2 Angstrom Resolution. Science 286: 255-260, 1999.
A. K. Dioumaev, L. S. Brown, R. Needleman and J. K. Lanyi. FTIR Spectra of a Late Intermediate of the Bacteriorhodopsin Photocycle Suggest Transient Protonation of Asp-212. Biochemistry 38: 10070-10078, 1999.
H. Luecke, B. Schobert, H. -T. Richter, J. -P. Cartailler and J. K. Lanyi. Structure of Bacteriorhodopsin at 1.55 Angstrom Resolution. J.Mol.Biol. 291: 899-911, 1999.
A. K. Dioumaev, L. S. Brown, R. Needleman and J. K. Lanyi. Partitioning of Free Energy Gain between the Photoisomerized Retinal and the Protein in Bacteriorhodopsin. Biochemistry 37: 9889-9893, 1998.
Structural Biology and Molecular Biophysics