BIOLOGICAL MEMBRANES

!             Overview  biological roles  structural features

!             Membrane lipids  general structures   aggregation states   polymorphism   thermal transitions   electrical conductivity   electrostatic effects   molecular dynamics (translational and rotational diffusion,

flip-flop) 

!             Membrane proteins  crystallization  overview of structural features  structure/function relations:

bacterial photosynthetic reaction centerbacteriorhodopsin

Biochemistry 585; Membrane ProteinsReading List

CRYSTALLIZATION

*C. Ostermeier & H. Michel, ACrystallization of membrane proteins@, Curr. Opinion Struct. Biol. 7, 697-701 (1997). #C. Ostermeier, S. Iwata, B. Ludwig & H. Michel, AFv fragment-

mediated crystallization of the membrane protein bacterial cytochrome c oxidase@, Nature Structural Biology, 2, 842-846 (1995). 

Optional: *E.M. Landau & J.P. Rosenbusch, ALipidic cubic phases: A novel concept for the crystallization of membrane proteins@, Proc. Natl. Acad. Sci. USA 93, 14532-14535 (1996).

[ *pdf files on website; #reprint will be provided ]

STRUCTURES AND FUNCTIONS

Overview: *S. Scarlata, "Membrane Protein Structure", Chap. 1, Section 2, Biophysical Society on-line textbook. *J.U. Bowie, "Membrane proteins: are we destined to repeat history", Curr. Opinion Struct. Biol. 10, 435-437 (2000).

*G.G. Shipley, "Lipids; Bilayers and non-bilayers: structures, forces and protein crystallization", Curr. Opinion Struct. Biol. 10, 471-473 (2000).

Electron Transfer Mechanisms:

Optional:*J.R. Winkler, "Electron tunneling pathways in proteins", Curr. Opinion in Chem. Biol. 4, 192-198 (2000).

#C. C. Page, C.C. Moser X. Chen & P.L. Dutton, "Natural engineering principles of electron tunneling in biological oxidation-reduction", Nature 402, 47-52 (1999).

Bacterial Photosynthetic Reaction Center: #U. Ermler, H. Michel & M. Schiffer, "Structure and function of the photosynthetic reaction center from Rhodobacter sphaeroides", J. Bioenerg. Biomembr. 26, 5-15 (1994).

*J.P. Allen & J.C. Williams, "Photosynthetic reaction centers", Minireview, FEBS Lett. 438, 5-9 (1998).

#N.W. Woodbury & J.P. Allen, APathway, kinetics and thermodynamics of electron transfer in wild type and mutant reaction centers of purple nonsulfur bacteria@, in Anoxygenic Photosynthetic Bacteria, R.E. Blankenship et al., eds, Chap. 24, pp. 527-557, Kluwer Acad. Publ., 1995.

Optional: *J. Deisenhofer et al., ACrystallographic refinement at 2.3 resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis@, J. Mol. Biol. 246, 429-457 (1995)].

*P.K. Fyfe and M.R. Jones, "Re-emerging structures: continuing crystallography of the bacterial reaction centre", Biochim. Biophys. Acta 1459, 413-421 (2000).

*M.Y. Okamura et al., "Proton and electron transfer in bacterial reaction centers", Biochim. Biophys. Acta 1458, 148-163 (2000).]

Bacteriorhodopsin: *J.K. Lanyi and H. Luecke “Bacteriorhodopsin”, Curr. Opinion Struct. Biol., 11, 415-419 (2001).

#W. Khlbrandt "Bacteriorhodopsin- the movie", Nature 406, 569-570 (2000). Optional: *J.K. Lanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3, Biophysical Society on-line textbook.

BIOLOGICAL ROLES OF MEMBRANES

SELECTIVE PERMEABILITY BARRIERS (CELL COMPARTMENTALIZATION): PUMPS, GATES SIEVES

STRUCTURAL ORGANIZATION OF CELLULAR PROCESSES (ENERGY TRANSDUCTION): RESPIRATION, PHOTOSYNTHESIS, VISION

RECEPTORS FOR EXTERNAL STIMULI: HORMONES, NEUROTRANSMITTERS

CELL RECOGNITION: IMMUNE RESPONSE, TISSUE FORMATION

INTERCELLULAR COMMUNICATION: NERVE IMPULSE TRANSMISSION

MOST MEMBRANES ARE MULTI-FUNCTIONAL

STRUCTURAL FEATURES OF MEMBRANES MULTIPLE COMPONENTS

LIPIDS (PHOSPHOLIPIDS, GLYCOLIPIDS, CHOLESTEROL):BILAYER STRUCTURE FORMS MAIN PERMEABILITY

BARRIER.PROTEINS (PERIPHERAL, INTEGRAL): PROVIDE BOTH

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS.CARBOHYDRATE (COVALENTLY BOUND TO LIPID AND

PROTEIN): SURFACE RECOGNITION. BROAD COMPOSITIONAL VARIABILITY

CORRELATED WITH FUNCTION MOSTLY SELF ASSEMBLING

HYDROPHOBIC AND ELECTROSTATIC FORCES LEAD TO BILAYER FORMATION AND PROTEIN INCORPORATION (CARBOHYDRATE ADDED ENZYMATICALLY AFTER ASSEMBLY)

ASYMMETRIC

INSIDE DIFFERENT FROM OUTSIDE WITH RESPECT TO LIPID AND PROTEIN (CARBOHYDRATE ONLY FOUND ON OUTER SURFACE)

DYNAMIC STRUCTUREFLUIDITY, FLEXIBILITY, TWO-DIMENSIONAL DIFFUSION

BIOLOGICAL SIGNIFICANCE OF LIPID POLYMORPHISM

POTENTIAL TO FORM NONBILAYER STRUCTURES MAY ALLOW DISCONTINUITIES IN BILAYER AND THEREBY PROMOTE:

MEMBRANE FUSION AND VESICLE FORMATION DURING CELL DIVISION.    VESICLE-MEDIATED PROTEIN TRAFFICKING.    INTEGRATION OF NON-LIPID COMPONENTS INTO MEMBRANE.    MOVEMENT OF MACROMOLECULES THROUGH MEMBRANE.    LATERAL MOVEMENT OF MACROMOLECULES.    STABILIZATION OF MEMBRANE PROTEIN COMPLEXES.    CONFORMATIONAL INTERCONVERSIONS ASSOCIATED WITH PROTEIN FUNCTION.

TRANSLATIONAL DIFFUSION IN MEMBRANES

USUALLY MEASURED BY FRAP (FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING) USING FLUOROPHORE-

LABELLED LIPIDS. INVOLVES PHOTOBLEACHING A SMALL REGION OF MEMBRANE SURFACE WITH LASER AND MEASURING TIME DEPENDENCE OF MOLECULAR DIFFUSION INTO BLEACHED AREA. Dtrans (translational diffusion coefficient) RELATED TO MEAN SQUARE DISPLACEMENT:

_r2 4 Dtrans t

FOR BOTH LIPIDS AND PROTEINS, Dtrans 10-8 cm2s-1 at 25 °C. THUS, IN 1 SECOND: _ r2 = 4 x 10-8 cm2

_(r2)1/2 (MEAN DISPLACEMENT) = 2 x 10-4 cm = 2 microns

(i.e. MOVEMENT IS RAPID).

MEASUREMENT OF MEMBRANE FLUIDITY AND MOLECULAR ROTATION BY FLUORESCENCE DEPOLARIZATIONUSE A COVALENTLY ATTACHED FLUOROPHORE, OR A FLUORESCENT PROBE WHICH PARTITIONS INTO THE BILAYER (e.g. DPH; DIPHENYLHEXATRIENE). EXCITE WITH POLARIZED LIGHT AND MEASURE POLARIZATION OF FLUORESCENCE. IF FLUOROPHORE ROTATES DURING EXCITED STATE LIFETIME,

FLUORESCENCE WILL BECOME DEPOLARIZED.

DEFINITIONS:P = POLARIZATION = (I - I) / (I + I)

r = ANISOTROPY = (I - I) / (I + 2I) PERRIN EQUATION:

r0 / r = DEGREE OF DEPOLARIZATION = 1 + (F / C)

 WHERE:

r0 = ANISOTROPY IN RIGID MATRIX (I.E. NO

ROTATION)r = ANISOTROPY IN MEMBRANE F = FLUORESCENCE LIFETIME

C = ROTATIONAL CORRELATION TIME = 1 / DROT

DROT = ROTATIONAL DIFFUSION COEFFICIENT

PERRIN EQUATION ALLOWS ROTATIONAL CORRELATION TIME TO BE DETERMINED. THIS CAN BE RELATED TO SOLVENT VISCOSITY (FOR A SPHERICAL MOLECULE) BY: 

c = V / k T

 where: 

= VISCOSITYV = VOLUME OF FLUOROPHORE

USUALLY USE A CALIBRATION CURVE TO CALCULATE MICROVISCOSITY OF MEDIUM. IN GENERAL:  lipid bilayer 100 water

 CAN ALSO BE APPLIED TO PROTEINS IN A MEMBRANE TO OBTAIN

DROT. FOR TWO-DIMENSIONAL ROTATIONAL MOTION:

Drot = k T / 4 a2 h

FOR A "TYPICAL" MEMBRANE PROTEIN: Drot = 105 s-1; c = 2 s

ha

ELECTROSTATIC EFFECTS AT MEMBRANE SURFACES MEMBRANE SURFACE CHARGE WILL INFLUENCE LOCAL CONCENTRATIONS OF CHARGED SPECIES, INCLUDING HYDROGEN IONS, SALT IONS AND PROTEINS. THE SURFACE POTENTIAL OF A MEMBRANE CAN BE CALCULATED FROM ELECTROSTATIC DOUBLE LAYER THEORY (GUOY-CHAPMAN THEORY; cf. CEVC & MARSH, “PHOSPHOLIPID BILAYERS”, WILEY-INTERSCIENCE, 1987).

(in mV) = (2kT/Ze) ln (0.36 Ac C1/2)Z = charge valency of counterionsAc = area per charge at membrane surface (in nm2)C = molar concentration of salt ions

FROM THIS POTENTIAL, ONE CAN CALCULATE THE LOCAL CONCENTRATION OF A CHARGED PROTEIN, AND THE LOCAL pH:

[P]surface = [P]bulk exp(-Z / kT) where Z is the net protein charge.pHsurface = pHbulk + e / 2.3 kT

NOTE THAT IS ALWAYS NEGATIVE FOR BIOMEMBRANES. ALSO, BOTH OF THESE QUANTITIES WILL BE STRONGLY AFFECTED BY SALT CONCENTRATION.

HIGH RESOLUTION MEMBRANE PROTEIN CRYSTAL STRUCTURES (as of March, 2002) [~17,000 soluble protein structures listed] 1- Porin: M.S. Weiss & G.E. Schulz, J. Mol. Biol. 227, 493-509 (1992). 2- Bacterial photosynthetic reaction center: J. Deisenhofer et al., J. Mol. Biol. 246, 429-457 (1995). 3- Prostaglandin synthase: D. Picot et al., Nature 367, 243-249 (1994). 4- Cytochrome c oxidases: S. Iwata et al., Nature 376, 660-669 (1995); T. Tsukihara et al., Science 272, 1136-1144 (1996); Soulimane et al., EMBO J. 19, 1766-76 (2000). 5- Bacterial light-harvesting complex: G. McDermott et al., Nature 374, 517-521 (1995); J. Koepke et al., Structure 4, 581-597 (1996). 6- -Hemolysin: L. Song et al., Science 274, 1859-1866 (1996). 7- Cytochrome bc1: D. Xia et al., Science 277, 60-66 (1997); Z. Zhang et

al., Nature 392, 677-684 (1998); S. Iwata et al., Science 281, 64-71 (1998); C. Hunte et al., Structure 8, 669-684 (2000). 

8- Bacteriorhodopsin: H. Luecke et al., J. Mol. Biol. 291, 899-911 (1999) 9- Potasssium ion channel: D.A. Doyle et al., Science 280, 69-77 (1998). 10- Iron transport protein (FhuA): A.D. Ferguson et al., Science 282, 2215-2220 (1998). 11- Mechanosensitive ion channel (MscL): G. Chang et al., Science 282, 2220-2226 (1999). 12- Fumarate reductase: T.M. Iverson et al., Science 284, 1961-1966 (1999); C.R.D. Lancaster et al., Nature 402, 377-385 (1999). 13- Outer membrane active transporter (FepA): S.K. Buchanan et al., Nature Struct. Biol. 6, 56-63 (1999). 14- Squalene-hopene cyclase: K.U. Wendt et al., J. Mol. Biol. 286, 175-187 (1999).

15- Outer membrane phospholipase A: H.J. Snijder et al., Nature 401, 717-721 (1999). 

16- Sarcoplasmic reticulum calcium pump: Toyoshima et al., Nature 405, 647-655 (2000). 17- E. coli glycerol channel: Fu et al. Science 290, 481-486 (2000). 18- Rhodopsin: Palczewski et al., Science 289, 739-745 (2000). 19- Halorhodopsin: Kolbe et al., Science 288, 1390-1396 (2000). 20- TolC outer membrane pore: Koronakis et al., Nature 405, 914-919 (2000). 21- Sensory Rhodopsin: Leucke et al., Science 293, 1499-1503 (2001); Royant et al., PNAS 98, 10131-10136 (2001). 22- Photosystem I: Jordan et al., Nature 411, 909-917 (2001); Barber, Nature Struct. Biol. 8, 577-579 (2001). 23- Photosystem II: Zouni et al., Nature 409, 739-743 (2001). 

24- C1C chloride channel: Dutzler et al., Nature 415, 287-294 (2002)

23- Formate dehydrogenase: Jormakka et al., Science 295, 1863-1869 (2002).

Web site: http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

CURRENT OPINION IN STRUCTURAL BIOLOGY, 7, 697-701 (1997).Crystallization of membrane proteinsChristian Ostermeier* and Hartmut Michel†

Five new membrane protein structures have beendetermined since 1995 using X-ray crystallography: bacteriallight-harvesting complex; bacterial and mitochondrialcytochrome c oxidases; mitochondrial bc 1 complex; anda-hemolysin. These successes are partly based on advancesin the crystallization procedures for integral membrane

proteins. Variation of the size of the detergent micelle and/orincreasing the size of the polar surface of the membraneprotein is the most important route to well-ordered membraneprotein crystals. The use of bicontinuous lipidic cubic phasesalso appears to be promising.Addresses* Department of Molecular Biophysics and Biochemistry, YaleUniversity, Bass Center 433, Whitney Avenue, New Haven,CT 06520-8114, USA; e-mail: osti@laplace.csb.yale.edu† Max-Planck-Institut fur Biophysik, Abteilung fur MolekulareMembranbiologie, Heinrich-Hoffmann-Strasse 7, 60528Frankfurt/Main, Germany; e-mail: michel@mpibp-frankfurt.mpg.de

CRYSTALLIZATION OF INTEGRAL MEMBRANE PROTEINS SOLUBILIZED IN DETERGENT MICELLES

CRYSTALS STABILIZED MAINLY BY POLAR INTERACTIONS BETWEEN PROTEIN MOLECULES AND BETWEEN DETERGENT MOLECULES.

DETERGENT MOLECULES MUST FIT INTO CRYSTAL LATTICE; THUS THEIR SIZE (SMALLER IS BETTER) AND CHEMISTRY ARE

IMPORTANT.

ADDITION OF SMALL AMPHIPHILES TO CRYSTALLIZATION MEDIUM OFTEN ENHANCES CRYSTAL FORMATION BY REPLACING THOSE DETERGENT MOLECULES THAT STERICALLY INTERFERE WITH LATTICE FORMATION. ALSO, BY MAKING MICELLES SMALLER, THEY CAN ALLOW BETTER CONTACT BETWEEN POLAR SURFACES OF PROTEIN.

SMALL AMPHIPHILES ALSO INCREASE PROTEIN SOLUBILITY.

SEE: NOLLER ET AL., FEBS LETT. 504, 179-186 (2001) FOR DISCUSSION OF MECHANISM OF CUBIC PHASE CRYSTALLIZATIONProc. Natl. Acad. Sci. USAVol. 93, pp. 14532–14535, December 1996

Lipidic cubic phases: A novel concept for the crystallization of membrane proteinsEHUD M. LANDAU AND JŰRG P. ROSENBUSCHBiozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, SwitzerlandCommunicated by H. Ronald Kaback, University of California, Los Angeles, CA, September 30, 1996 (received for review August 12, 1996)

ABSTRACT Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishingthis by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolidlipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (‘‘seeding’’) and support growth by lateral diffusion of protein molecules in the membrane (‘‘feeding’’). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution (NOW TO 1.6 ), with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; = = 90º and = 120º.

(HALORHODOPSIN ALSO CRYSTALLIZED IN THIS WAY.)

PNAS 96, 14706-14711 (1999)Structural details of an interaction between cardiolipin and an integral membrane proteinKatherine E. McAuley* , Paul K. Fyfe‡ , Justin P. Ridge‡ , Neil W. Isaacs† , Richard J. Cogdell*, and Michael R. Jones‡

*Division of Biochemistry and Molecular Biology and † Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ, United Kingdom; and ‡ Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2UH, United KingdomEdited by Johann Deisenhofer, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 27, 1999 (received for review May 3, 1999)

Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid–protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data col-lected over the resolution range 30.0–2.1 Å show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral in-tramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the imme-diate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.

PRINCIPLES OF MEMBRANE PROTEIN STRUCTURE [Scarlata, "Membrane Protein Structure"; see also: White & Wimley, Ann. Rev. Biophys. Biomol. Struct. 28, 319 (1999); White, in “Membranes”, Biophysical Society on-line textbook].

   MEMBRANE PROTEIN ENVIRONMENT IS COMPLEX; IT INVOLVES THE AQUEOUS REGION OUTSIDE MEMBRANE, ELECTRICAL CHARGES AT THE MEMBRANE SURFACE, AND THE HYDROPHOBIC INTERIOR OF THE MEMBRANE. THE STEEP DIELECTRIC GRADIENT MAKES IT UNFAVORABLE TO BURY A CHARGE (20 kCAL/MOLE) OR HAVE AN UNSATISFIED H-BOND (5 kCAL/MOLE); CONTROLS WHICH RESIDUES INCORPORATE WITHIN THE MEMBRANE AND WHICH REMAIN OUTSIDE, AS WELL AS SECONDARY AND TERTIARY FOLDING (-HELICES AND -SHEETS FAVORED; LOOPS AND RANDOM COILS DISFAVORED).

LIPID HEAD GROUPS CAN HAVE STRONG ELECTROSTATIC AND H-BONDING INTERACTIONS WITH INTERFACIAL RESIDUES OF A MEMBRANE PROTEIN.

   HYDROPHOBIC THICKNESS OF THE BILAYER MUST MATCH THE HYDROPHOBIC LENGTH OF THE PROTEIN, e.g. TRANSMEMBRANE HELIX MUST BE 18 RESIDUES LONG. BILAYER THICKNESS MAY STABILIZE CERTAIN CONFORMATIONAL STATES.

    HYDROCARBON CHAIN PACKING MAY ALSO STABILIZE CERTAIN PROTEIN STRUCTURES; FAVORS COMPONENTS WHICH DO NOT GREATLY DISRUPT THEIR INTERACTIONS; e.g., PROTEIN CYLINDRICAL SHAPES ARE PREFERRED.

    SOME GENERALIZATIONS: TERTIARY STRUCTURES OF MEMBRANE PROTEINS HAVE SIMILAR PACKING AS SOLUBLE PROTEINS; HELICES TILTED 20 TO ALLOW PACKING BETWEEN SIDE CHAINS; H-BONDS BETWEEN HELICES ARE RARE AND SALT BRIDGES NOT FOUND. BECAUSE OF HELIX DIPOLES, ANTIPARALLEL ARRANGEMENT OF TRANSMEMBRANE HELICES PREFERRED. TRP AND TYR MAINLY PRESENT AT INTERFACES; ACT AS "ANCHORS".

PROSTAGLANDIN H2 SYNTHASE-1

INTEGRAL MEMBRANE PROTEIN, LOCATED PRIMARILY IN THE ENDOPLASMIC RETICULUM.

CATALYZES THE FIRST COMMITTED STEP IN PROSTAGLANDIN BIOSYNTHESIS (ARACHIDONATE TO PROSTAGLANDIN H2).

BIFUNCTIONAL: CYCLOOXYGENASE (TARGET FOR NSAID’S: ASPIRIN, IBUPROFEN, INDOMETHACIN); PEROXIDASE

ANCHORED TO ONE LEAFLET OF BILAYER BY AMPHIPATHIC HELICES.

PORINS

FOUND IN OUTER MEMBRANES OF GRAM-NEGATIVE BACTERIA.

FORM WATER-FILLED CHANNELS THAT ALLOW THE INFLUX/OUTFLUX OF SMALL HYDROPHILIC MOLECULES.

HAVE TRIMERIC, BETA-BARREL STRUCTURES; RESIDUES ALTERNATE BETWEEN FACING INWARD AND OUTWARD. THUS, DO NOT HAVE LONG STRETCHES OF HYDROPHOBIC RESIDUES, AS IN TRANSMEMBRANE HELICES.

PORES NARROWED BY INWARD FOLDING OF A LOOP INTO LUMEN OF BARREL. HAVE WIDE ENTRANCE AND WIDE EXIT, AND A SHORT CENTRAL CONSTRICTION (ABOUT 10 DEEP AND 10 WIDE). MINIMIZES FRICTIONAL CONTACT WITH WALLS, WHILE STILL EXCLUDING LARGE MOLECULES.

A SIMPLIFIED OVERVIEW OF ELECTRON TRANSFER THEORY

ELECTRON TRANSFER (ET) IS A FUNDAMENTAL PROCESS IN BIOLOGY, OCCURRING WITHIN AND BETWEEN PROTEIN MOLECULES WHICH SERVE AS SCAFFOLDING FOR A VARIETY OF REDOX CENTERS (METAL IONS, PORPHYRINS, FLAVINS, QUINONES, ETC.).

AMONG THE KEY QUESTIONS ARE:1- HOW DO ELECTRONS MOVE OVER THE SOMETIMES LONG DISTANCES BETWEEN REDOX CENTERS WHICH ARE IMPOSED BY THE PROTEIN MATRIX (i.e. PATHWAYS)? 2- HOW DO DISTANCES BETWEEN REDOX CENTERS, FREE ENERGY CHANGES FOR THE ET PROCESS, AND "SOLVENT" ENVIRONMENTS OF THE REDOX CENTERS INFLUENCE ET RATES? 3- HOW DOES THE INTERVENING PROTEIN MATRIX INFLUENCE ET RATES?

THE BACTERIAL PHOTOSYNTHETIC REACTION CENTER HAS BECOME AN IMPORTANT MODEL SYSTEM FOR INVESTIGATING THESE QUESTIONS (ITS STRUCTURE IS KNOWN, IT CONTAINS 8 REDOX CENTERS WHICH SPAN A DISTANCE OF APPROXIMATELY 80Å, AND ITS KINETIC PROPERTIES SPAN A TIME RANGE FROM PICOSECONDS TO TENS OF SECONDS).

THE STARTING POINT FOR THEORETICAL TREATMENTS OF ET REACTIONS IS THE FOLLOWING EQUATION (OBTAINED FROM TIME-DEPENDENT QUANTUM MECHANICAL PERTURBATION THEORY): kET = (4π2/h) VAB

2 FC

 WHERE VAB IS THE MATRIX ELEMENT FOR ELECTRONIC COUPLING

BETWEEN THE TWO REDOX SITES AND FC IS THE FRANCK-CONDON (NUCLEAR) FACTOR.

VAB IS PROPORTIONAL TO THE OVERLAP OF THE ELECTRONIC

WAVEFUNCTIONS OF THE DONOR AND ACCEPTOR, AND IS THE PRINCIPAL ORIGIN OF THE DISTANCE DEPENDENCE OF ET (ALSO PROVIDES A ROLE FOR THE INTERVENING PROTEIN MATRIX).

SIMPLEST MODEL (NEGLECTING ROLE OF INTERVENING MEDIUM) PREDICTS VAB PROPORTIONAL TO exp (-αR). APATHWAYS@

CONCEPT PROPOSES THAT ELECTRONS TUNNEL BETWEEN LOCALIZED REDOX CENTERS, UTILIZING BOTH THROUGH-BOND AND THROUGH-SPACE ROUTES WHICH ARE HIGHLY SENSITIVE TO MOLECULAR STRUCTURE (METHODS FOR CALCULATING THE EFFECTIVENESS OF THESE ROUTES HAVE BEEN DEVELOPED).

FC ORIGINATES FROM THE REQUIREMENT (FRANCK-CONDON PRINCIPLE) THAT THE NUCLEAR CONFIGURATION OF THE REACTANTS MUST BE SUCH THAT THE ENERGY OF THE REACTANTS AND PRODUCTS ARE EQUAL AT THE TRANSITION STATE (THIS OCCURS VIA THERMAL FLUCTUATIONS AND/OR VIBRATIONS; THIS PROVIDES A ROLE FOR PROTEIN DYNAMICS; ENERGY TO ACHIEVE THIS CALLED "REORGANIZATION ENERGY"), I.E. ET OCCURS BETWEEN STATES WHOSE NUCLEAR COORDINATES DO NOT CHANGE. REORGANIZATION ENERGY IS OFTEN DIVIDED BETWEEN CHANGES OCCURRING AT REDOX CENTER (INNER SPHERE) AND THOSE OCCURRING IN SURROUNDING PROTEIN/WATER MATRIX (OUTER SPHERE).

FC FACTOR CONTAINS THE DEPENDENCE OF ET RATE ON THE FREE ENERGY CHANGE BETWEEN REACTANTS AND PRODUCTS AND ON THE REORGANIZATION ENERGY.

THE SIMPLEST THEORETICAL TREATMENT OF FC FACTORS IS DUE TO MARCUS (USING A CLASSICAL HARMONIC OSCILLATOR MODEL, WHICH GENERATES PARABOLIC POTENTIAL ENERGY CURVES). YIELDS THE WIDELY USED MARCUS EQUATION: kET = (4π2/h) VAB

2 [1/(4πλkT)1/2] exp [-(λ + ΔG0)2/4λkT]

 WHERE λ = REORGANIZATION ENERGY. ΔG0 = -RT ln Keq = -n F E0

 

WHERE F = FARADAY CONSTANT = 23.09 kcal/volt ΔGI = (λ + ΔG0)2 / 4λ

THE RELATIONSHIP BETWEEN kET AND ΔG0 IS SHOWN

SCHEMATICALLY IN FOLLOWING GRAPHS. THIS YIELDS THE FOLLOWING PICTURE: 

AS THE DRIVING FORCE FOR ET INCREASES, THE RATE INCREASES AND THE ACTIVATION ENERGY DECREASES. WHEN λ = ΔG0, kET REACHES A MAXIMUM AND THE ACTIVATION ENERGY

BECOMES ZERO. FURTHER INCREASES IN DRIVING FORCE RESULT IN A DECREASE IN REACTION RATE AND AN INCREASE IN ACTIVATION ENERGY (MARCUS INVERTED REGION).

ALTHOUGH MORE SOPHISTICATED TREATMENTS OF FC FACTORS EXIST, OUR UNDERSTANDING OF THE WAYS IN WHICH EXPERIMENTAL VARIABLES INFLUENCE REACTION RATES, ESPECIALLY IN PROTEINS, IS OFTEN NOT SUFFICIENT TO JUSTIFY USE OF MORE RIGOROUS THEORETICAL MODELS. THUS, FOR EXAMPLE, TEMPERATURE CAN AFFECT ELECTRONIC COUPLING, DRIVING FORCE, AND REORGANIZATIONAL ENERGY; SEVERAL VIBRATIONAL MODES MAY BE COUPLED TO THE ET STEP; ETC.

BACTERIAL PHOTOSYNTHETIC REACTION CENTER (R. viridis)

CRYSTALLIZATION:AMMONIUM SULFATE PRECIPITATION IN PRESENCE OF LDAO AND HEPTANE-1,2,3-TRIOL.

STRUCTURE:SUBUNITS (FOUR): L, M, H PLUS TIGHTLY-BOUND 4-HEME

CYTOCHROME (c-TYPE; ABSENT IN SOME RC’S).

COFACTORS:BOUND BY L AND M SUBUNITS;4 BCHL, 2 BPHE, 2 QUINONES (QB SITE ONLY PARTLY

OCCUPIED),1 CAROTENOID, 1 Fe (II);CAROTENOID AND QUINONE STRUCTURES VARY WITH

SPECIES.

LIPIDS:1 LDAO WELL-ORDERED IN CRYSTAL (NEAR QA BINDING

SITE);CYTOCHROME SUBUNIT HAS A COVALENTLY LINKED

DIGLYCERIDE (TO SULFUR OF C-TERMINAL CYSTEINE) WHICH EXTENDS INTO THE MEMBRANE.

FUNCTION:MECHANISM OF ELECTRON TRANSFER

ELECTRON FLOW ASYMMETRIC (ONLY VIA L PATHWAY).

PHOTON ABSORPTION (OR ENERGY TRANSFER) RAISES P TO FIRST EXCITED SINGLET STATE (P*; NATURAL LIFETIME ~ 3 ns)

ELECTRON TRANSFER TO Bph (HA) OCCURS IN ~ 3.5 ps; ROLE OF BRIDGING Bchl (BA) UNCERTAIN (i.e. DOES ELECTRON RESIDE HERE FOR ANY FINITE TIME, OR IS BA ONLY INVOLVED IN COUPLING BETWEEN P AND HA?). IT CLEARLY PLAYS A ROLE, SINCE RATE IS TOO FAST FOR DIRECT TRANSFER FROM P TO HA. ELECTRON TRANSFER TO QA OCCURS IN ~ 200 ps.

ELECTRON TRANSFER TO QB OCCURS IN ~ 100 μs (Fe DOES NOT PLAY A DIRECT ROLE IN TRANSFER; EXACT FUNCTION UNCLEAR; MAY BE STRUCTURAL).

QB PICKS UP TWO ELECTRONS AND TWO PROTONS AND DISSOCIATES FROM BINDING SITE (~ 5 ms; THIS IS RATE-LIMITING STEP); SITE REFILLED FROM QUINONE POOL.

REDUCED QB IS REOXIDIZED BY CYTOCHROME bc1; ELECTRONS THEN TRANSFERRED TO CYTOCHROME c2 AND THEN TO P+ (EITHER VIA 4-HEME CYTOCHROME OR DIRECTLY).

OVERALL QUANTUM EFFICIENCY IS CLOSE TO UNITY.

ROLE OF PROTEIN: GENERALLY THOUGHT THAT PROTEIN MOTIONS ARE COUPLED TO ELECTRON TRANSFER (e.g. VIBRATIONAL MODES; RELAXATIONS THAT STABILIZE VARIOUS INTERMEDIATE STATES).

PROTON TRANSPORT IN BACTERIORHODOPSIN

SIMPLEST KNOWN EXAMPLE OF A TRANSMEMBRANE ION PUMP.

DARK ADAPTED STATE

2 CHANNELS LEAD FROM ACTIVE SITE TO SURFACE: EXTRACELLULAR (HYDROPHILIC, WIDE); CONTAINS H-BONDED NETWORK OF FOUR RESIDUES (ARG-82, TYR-57, GLU-194, GLU-204), AND AT LEAST SIX BOUND WATER MOLECULES.

CYTOPLASMIC (HYDROPHOBIC, NARROW); CONTAINS ONLY ONE RESIDUE INVOLVED IN PROTON TRANSPORT (ASP-96; HAS AN UNUSUALLY HIGH pK), AND FEWER BOUND WATERS. TO REPROTONATE THE SCHIFF BASE DURING THE PHOTOCYCLE, THIS REGION HAS TO UNDERGO CONFORMATIONAL CHANGES, ALLOWING WATER TO ENTER.

ACTIVE SITE CAN BE THOUGHT OF AS CONSISTING OF A HIGHLY POLARIZED WATER MOLECULE (W402) COORDINATED BY THE PROTONATED RETINAL SCHIFF BASE, WHICH IS SALT-LINKED TO TWO ANIONIC ASP RESIDUES, ASP-85 AND ASP-212. SCHIFF BASE IS DEPROTONATED DURING THE PHOTOCYCLE.

EVENTS FOLLOWING LIGHT ABSORPTION

  RETINAL PHOTOISOMERIZATION IS COUPLED TO PROTEIN CONFORMATION CHANGES. THIS IS THE RESULT OF A STERIC AND ELECTROSTATIC CONFLICT OF THE CHROMOPHORE WITH ITS BINDING SITE. RELAXATION OF THIS CONFLICT DRIVES THE THERMAL REACTIONS OF THE PHOTOCYCLE.

PROTON IS TRANSFERRED TO ASP-85 WITHIN ABOUT 50 S. PROTON MAY BE DERIVED FROM SCHIFF BASE (SUGGESTED THAT SCHIFF BASE pK DECREASES AND ASP pK INCREASES DUE TO CHANGES IN ENVIRONMENT: SCHIFF BASE N MOVES TO HYDROPHOBIC REGION AND H-BONDS FORM TO CARBOXYL GROUP); MAY BE HELPED BY A SMALL MOVEMENT OF HELIX C WHICH BRINGS THEM CLOSER TOGETHER. ALSO POSSIBLE THAT PROTON DERIVES FROM THE BOUND WATER MOLECULE, GENERATING HYDROXYL WHICH REMOVES PROTON FROM RETINAL.

PROTONATION STATES OF ASP-85, GLU-204 AND GLU-194 LINKED. TRANSFER OF PROTON TO ASP-85 CAUSES MOVEMENT OF ARG-82 TOWARDS BOTTOM OF CHANNEL. THIS CAUSES pK OF GLU-204 TO DECREASE; GLU-204 TRANSFERS PROTON TO GLU-194, WHICH RELEASES PROTON AT EXTRACELLULAR SURFACE.

PROTEIN CONFORMATION CHANGE IN M INTERMEDIATE IS CAUSED BY RETINAL STRAIGHTENING; 13-METHYL PUSHES ON TRP-182, MOVING HELIX F. CAUSES pK OF ASP-96 TO DECREASE, DUE TO INCREASE IN HYDRATION OF CYTOPLASMIC CHANNEL; RESULTS IN PROTON TRANSFER TO SCHIFF BASE. DARK RE-ISOMERIZATION OF RETINAL CAUSES REVERSAL OF PROTEIN CONFORMATIONAL CHANGE. RESTORES THE HIGH pK OF ASP-96, LEADING TO REPROTONATION FROM CYTOPLASM. THIS CAUSES PROTON TRANSFER FROM ASP- 85 TO GLU-204 (VIA ARG-82 AND BOUND WATER MOLECULE), THEREBY COMPLETING THE PHOTOCYCLE.

SUMMARY OF OVERALL MECHANISM

PROTON TRANSPORT OCCURS VIA ALTERNATING ACCESS BETWEEN SCHIFF BASE AND THE TWO MEMBRANE SURFACES. DIRECTION OF TRANSFER IS CONTROLLED BY pK CHANGES CAUSED BY COUPLING BETWEEN RETINAL PHOTOISOMERIZATION AND PROTEIN CONFORMATIONAL CHANGES.

REPROTONATION OF SCHIFF BASE FROM CYTOPLASM REQUIRES THAT pK OFASP-96 BE LOWERED AND PROTON PATHWAY CREATED (PROBABLY VIA BOUND WATER).