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UCSD BIBC 100 - Structural Changes in Bacteriorhodopsin

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Structural Changes inBacteriorhodopsin During IonTransport at 2 AngstromResolutionHartmut Luecke,1,2,3* Brigitte Schobert,2,3Hans-Thomas Richter,2,3Jean-Philippe Cartailler,1,3Janos K. Lanyi2,3*Crystal structures of the Asp96to Asn mutant of the light-driven proton pumpbacteriorhodopsin and its M photointermediate produced by illumination atambient temperature have been determined to 1.8 and 2.0 angstroms reso-lution, respectively. The trapped photoproduct corresponds to the late M statein the transport cycle—that is, after proton transfer to Asp85and release of aproton to the extracellular membrane surface, but before reprotonation of thedeprotonated retinal Schiff base. Its density map describes displacements ofside chains near the retinal induced by its photoisomerization to 13-cis,15-antiand an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKavalues(where Kais the acid constant) of the Schiff base and Asp85. The structuralchanges detected suggest the means for conserving energy at the active siteand for ensuring the directionality of proton translocation.Active transport of ions against their electro-chemical potentials across cell and organellemembranes is carried out by proteins that cou-ple an energy-yielding reaction, such as hydrol-ysis of adenosine triphosphate, electron trans-fer, or isomerization of retinal, to the transmem-brane movement of ions. The mechanism ofthis kind of transport is a major, still unsolved,problem of membrane bioenergetics. A satis-factory mechanism will explain how the freeenergy gain from the driving reaction is usedto change, in an ordered way, the affinities ofbinding site(s) for the transported ion andhow the alternating accessibility of these ionbinding site(s) to the two membrane surfacesis mediated. Crystallographic structures areavailable for only a few transport proteins,and much effort is expended to improve theirresolutions to give unambiguous answers tothese questions.Bacteriorhodopsin is a light-driven protonpump, energized by the all-trans to 13-cis pho-toisomerization of the retinal chromophore (1).This small (26 kD) protein consists of seventransmembrane helices, A through G, and shortextramembrane segments (2–5). The structureof the unphotolyzed (BR) state, determinedmost recently at 1.55 Å resolution (6), revealeda three-dimensional interconnected hydrogen-bonded network of protein residues and watermolecules between the centrally located proton-ated retinal Schiff base and the extracellularsurface and provided clues about how release ofa proton to the extracellular membrane surfacemight be coupled to deprotonation of the Schiffbase. The local ␲-bulge distortion of helix G,with hydrogen bonding of two main-chainC⫽O groups to water molecules (6 ), suggested,in turn, that translocation of a proton from thecytoplasmic surface to the Schiff base might belinked to the shift of the protein conformationevident from low-resolution difference maps (7,8). Understanding how these processes causethe unidirectional translocation of a proton re-quires description of the structural changes inthe J, K, L, M, N, and O intermediates (9)ofthetransport cycle. The overall structure of theprotein and the sequence of proton transfers thatadd up to the full translocation of a proton fromone side of the membrane to the other areshown in Fig. 1. The crucial intermediate is theM state, because in M the Schiff base is de-protonated, Asp85to its extracellular side isprotonated, and a proton has been released tothe surface, but Asp96to its cytoplasmic sidehas not yet become deprotonated—that is, thisstate is after step 2 but before step 3 in Fig. 1.From spectroscopic and kinetic measure-ments of wild-type and mutant bacteriorho-dopsins, several substates of the M interme-diate have been identified and distinguishedby their accessibilities to the two membranesurfaces. In the early M state or states, theproton connectivity of the Schiff base is tothe extracellular side, whereas in the late Mstate(s) it is to the cytoplasmic side (10, 11).Transition of M1to M2(12) is associatedwith proton release to the extracellular sur-face (10, 13). Under physiological condi-tions, the pH is well above the pKa(where Kais the acid constant) for proton release, mak-ing the M1to M2reaction unidirectional andshifting the protonation equilibrium betweenAsp85and the Schiff base in the kineticscheme (L 7 M1) fully toward deprotonationof the Schiff base (that is, L 7 M13 M2).There is some uncertainty about the timingand the nature of structural changes in the pho-tocycle. From low-resolution difference mapsin projection, it is evident that large-scalechanges of helices F and G at the cytoplasmicsurface occur in the late M state (7, 14 ). Incontrast, large changes of amide I bands inFourier transform infrared spectroscopy (FTIR)difference spectra (15), and displacement ofhelix F as measured by spin-spin coupling oflabels on cytoplasmic interhelical loops (16),are seen only in the N state. In the Asp96to Asn(D96N) mutant, reprotonation of the Schiffbase is greatly slowed, and the late M state1Department of Molecular Biology and Biochemistry,2Department of Physiology and Biophysics,3UCI Pro-gram in Macromolecular Structure, University of Cal-ifornia, Irvine, CA 92697, USA.*To whom correspondence should be addressed. E-mail: [email protected] or [email protected] 1. Overall view of bacteriorhodopsin,shown with the retinal (purple) and residuesdirectly implicated in proton transport. Top,Cytoplasmic side. Arrows indicate proton trans-fer steps during the photochemical cycle. Num-bers refer to the sequential order: (1) deproto-nation of the Schiff base, protonation of Asp85;(2) proton release to the extracellular surface;(3) reprotonation of the Schiff base, deproto-nation of Asp96; (4) reprotonation of Asp96from the cytoplasmic surface; and (5) deproto-nation of Asp85, reprotonation of the protonrelease site. Coordinates are from (6).R ESEARCH A RTICLEwww.sciencemag.org SCIENCE VOL 286 8 OCTOBER 1999 255(termed MN) contains the changed amide Ibands of N (17). Although MNmight havesome features unique to the D96N mutant (18),at 3 Å resolution and in projection its structureis indistinguishable from M of the wild-typeprotein (14 ). The MNstate of D96N is easier totrap in a photostationary state than M of thewild type because the much slower decay al-lows virtually full conversion of


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