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BIOLOGICAL MEMBRANES

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r = ANISOTROPY = (I - I ) / (I 2I ) PERRIN EQUATION: r0 / r = DEGREE OF DEPOLARIZATION = 1 ... ANISOTROPY IN RIGID MATRIX (I.E. NO ROTATION) r = ANISOTROPY ... – PowerPoint PPT presentation

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Title: BIOLOGICAL MEMBRANES


1
BIOLOGICAL MEMBRANES !             Overview Ø 
biological roles Ø  structural
features !             Membrane lipids Ø 
general structures Ø  aggregation states Ø 
polymorphism Ø  thermal transitions Ø 
electrical conductivity Ø  electrostatic
effects Ø  molecular dynamics (translational
and rotational diffusion, flip-flop)   !        
     Membrane proteins Ø  crystallization Ø 
overview of structural features Ø 
structure/function relations bacterial
photosynthetic reaction center bacteriorhodopsin
2
Biochemistry 585 Membrane Proteins Reading List
CRYSTALLIZATION C. Ostermeier H. Michel,
ACrystallization of membrane proteins_at_, 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_at_, 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_at_, Proc. Natl. Acad. Sci. USA
93, 14532-14535 (1996). pdf files on website
reprint will be provided
3
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).

4
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).
5
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_at_, in Anoxygenic Photosynthetic Bacteria,
R.E. Blankenship et al., eds, Chap. 24, pp.
527-557, Kluwer Acad. Publ., 1995.
6
Optional J. Deisenhofer et al.,
ACrystallographic refinement at 2.3 D resolution
and refined model of the photosynthetic reaction
centre from Rhodopseudomonas viridis_at_, 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).
7
Bacteriorhodopsin J.K. Lanyi and H. Luecke
Bacteriorhodopsin, Curr. Opinion Struct. Biol.,
11, 415-419 (2001). W. K?hlbrandt
"Bacteriorhodopsin- the movie", Nature 406,
569-570 (2000).   Optional J.K. Lanyi
Bacteriorhodopsin, Bioenergetics, Chap. 3,
Biophysical Society on-line textbook.
8
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
9
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 STRUCTURE
  • FLUIDITY, FLEXIBILITY, TWO-DIMENSIONAL DIFFUSION

10
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.

11
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).

12
MEASUREMENT OF MEMBRANE FLUIDITY AND
MOLECULAR ROTATION BY FLUORESCENCE DEPOLARIZATION
  • USE 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

13
  • 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 
  • ? VISCOSITY
  • V 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

h
a
14
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 counterions Ac 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

Psurface Pbulk 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.
15
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). 
16
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). 
17
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)
18
23- Formate dehydrogenase Jormakka et al.,
Science 295, 1863-1869 (2002). Web site
http//blanco.biomol.uci.edu/Membrane_Proteins_x
tal.html
19
CURRENT OPINION IN STRUCTURAL BIOLOGY, 7, 697-701
(1997). Crystallization of membrane
proteins Christian Ostermeier and Hartmut
Michel Five new membrane protein structures
have been determined since 1995 using X-ray
crystallography bacterial light-harvesting
complex bacterial and mitochondrial cytochrome c
oxidases mitochondrial bc 1 complex
and a-hemolysin. These successes are partly based
on advances in the crystallization procedures for
integral membrane proteins. Variation of the size
of the detergent micelle and/or increasing the
size of the polar surface of the membrane protein
is the most important route to well-ordered
membrane protein crystals. The use of
bicontinuous lipidic cubic phases also appears to
be promising. Addresses Department of Molecular
Biophysics and Biochemistry, Yale University,
Bass Center 433, Whitney Avenue, New Haven, CT
06520-8114, USA e-mail osti_at_laplace.csb.yale.edu
Max-Planck-Institut fur Biophysik, Abteilung
fur Molekulare Membranbiologie,
Heinrich-Hoffmann-Strasse 7, 60528 Frankfurt/Main,
Germany e-mail michel_at_mpibp-frankfurt.mpg.de
20
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.

21
SEE NOLLER ET AL., FEBS LETT. 504, 179-186
(2001) FOR DISCUSSION OF MECHANISM OF CUBIC
PHASE CRYSTALLIZATION Proc. Natl. Acad. Sci.
USA Vol. 93, pp. 1453214535, December
1996 Lipidic cubic phases A novel concept for
the crystallization of membrane proteins EHUD M.
LANDAU AND JURG P. ROSENBUSCH Biozentrum,
University of Basel, Klingelbergstrasse 70,
CH-4056 Basel, Switzerland Communicated 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
accomplishing this 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
quasisolid lipidic 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.)
22
PNAS 96, 14706-14711 (1999) Structural details of
an interaction between cardiolipin and an
integral membrane protein Katherine 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 Kingdom Edited
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 lipidprotein 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 30.02.1 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.
23
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.
Ø   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.

24
Ø   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.
Ø    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.
Ø    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".
25
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
    NSAIDS ASPIRIN, IBUPROFEN, INDOMETHACIN)
    PEROXIDASE
  • ANCHORED TO ONE LEAFLET OF BILAYER BY
    AMPHIPATHIC HELICES.

26
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.

27
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).
28
THE STARTING POINT FOR THEORETICAL TREATMENTS OF
ET REACTIONS IS THE FOLLOWING EQUATION (OBTAINED
FROM TIME-DEPENDENT QUANTUM MECHANICAL
PERTURBATION THEORY)   kET (4p2/h) VAB2
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 (-aR).
APATHWAYS_at_ 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).
29
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.
30
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 (4p2/h) VAB2
1/(4p?kT)1/2 exp -(? ?G0)2/4?kT   WHERE ?
REORGANIZATION ENERGY.   ?G0 -RT ln Keq -n F
E0   WHERE F FARADAY CONSTANT 23.09
kcal/volt   ?GI (? ?G0)2 / 4?
31
THE RELATIONSHIP BETWEEN kET AND ?G0 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 ? ?G0, 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.
32
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 RCS).
  • 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.

33
  • 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).

34
  • 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).

35
PROTON TRANSPORT IN BACTERIORHODOPSIN
SIMPLEST KNOWN EXAMPLE OF A TRANSMEMBRANE ION
PUMP.
DARK ADAPTED STATE
  • 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.
  • 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.

36
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.


37
  • REPROTONATION OF SCHIFF BASE FROM CYTOPLASM
    REQUIRES THAT pK OFASP-96 BE LOWERED AND PROTON
    PATHWAY CREATED (PROBABLY VIA BOUND WATER).
  • 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.
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