BIOC. 585: BIOLOGICAL MEMBRANES
!
Overview
 biological roles
 structural features
!
Membrane lipids
 general structures
 aggregation states; polymorphism
 thermal transitions
 electrostatic effects
 molecular dynamics (translational and rotational diffusion,
flip-flop)
!
Membrane proteins
 crystallization
 overview of structural features
 structure/function relations:
photosynthetic electron transfer (cyt b6f)
ion transport (K+ channels)
bacteriorhodopsin
READING LIST
Biochem. 585: Membrane Proteins Reading List [all articles in pdf files on course website]
MEMBRANE PROTEIN CRYSTALLIZATION
Ostermeier & Michel, “Crystallization of membrane proteins”, Curr. Op. Struct. Biol. 7, 697-701 (1997).
Hunte & Michel, “Crystallization of membrane proteins mediated by antibody fragments”, Curr. Op.Struct.
Biol. 12, 503-508 (2002).
STRUCTURES AND FUNCTIONS
Overview
Scarlata, "Membrane Protein Structure", Chap. 1, Section 2, Biophysical Soc. on-line textbook.
Byrne & Iwata, “Membrane protein complexes”, Curr. Opinion in Struct. Biol. 12, 239-243 (2002).
Shipley, "Lipids; Bilayers and non-bilayers: structures, forces and protein crystallization", Curr. Op. Struct.
Biol. 10, 471-473 (2000).
Electron Transfer in Photosynthesis
Optional: Golbeck, “Photosynthetic Reaction Centers”, Biophysical Soc. on-line textbook.
Kurisu et al., “Structure of the cytochrome b6f complex of oxygenic photosynthesis” Science 302, 10091014 (2003).
Stroebel et al., “An atypical heme in the cytochrome b 6f complex”, Nature 426, 413-418 (2003).
Khlbrandt “Dual approach to a light problem”, Nature 426, 399-400 (2003)
Ion Channels
Doyle et al. “The structure of the potassium channel: molecular basis of K + conduction and selectivity”,
Science 280, 69-77 (1998).
Rees et al. “Crystallographic analysis of ion channels: lessons and challenges” J. Biol. Chem. 275, 713716 (2000).
MacKinnon “Potassium channels” FEBS Letters 555, 62-65 (2003).
Jiang et al. “X-ray structure of a voltage-dependent K+ channel”, Nature 423, 33-41 (2003).
Bacteriorhodopsin
Lanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3, Biophysical Soc. on-line textbook.
Optional: Neutze et al. “Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport”
Biochim. Biophys. Acta 1565, 144-167 (2002).
BIOLOGICAL ROLES OF MEMBRANES
~1/3 of all gene products in higher eukaryotes
are membrane proteins
 selective permeability barriers (cell compartmentalization):
pumps, gates, sieves
 structural organization of cellular processes (e.g. 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 enzymically 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
STRUCTURES OF MEMBRANE LIPIDS
LIPID POLYMORPHISM
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.
transport of macromolecules through
membrane.
lateral movement of macromolecules.
stabilization of membrane protein complexes.
conformational interconversions associated
with protein function.
MEMBRANE DYNAMICS
ELECTROSTATICS
TRANSLATIONAL (LATERAL) 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 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 / 6Drot
Drot = rotational diffusion coefficient
for a completely rigid system:
r0 = 0.33 (when absorption and emission
dipoles are at 90°)
r0 = 0.5 (when dipoles are parallel).
rotational diffusion coefficient is proportional to solvent
viscosity:
Drot = kT/frot = kT/ 6  V (for spherical molecule)
where:
frot = rotational frictional coefficient
 = viscosity
V = volume of fluorophore
via Perrin equation, rotational correlation time can be related
to solvent viscosity (for a spherical molecule) by:
c = V / k T
usually use a calibration curve to calculate microviscosity of
medium. in general:
 lipid bilayer  100  water
c can be determined directly by measuring time dependence
of anisotropy.
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 cylinder)
for a "typical" membrane protein: Drot = 105 s-1; c = 2 s
h
a
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 (Goüy-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 = surface charge density; area per charge at 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
STRUCTURES
WEB SITE:
blanco.biomol.uci.edu/Membrane_proteins_xtal.html
79 structures in pdb data base (as of March, 2004)
X-ray structures of membrane proteins (as of Jan. 2004)
24
22
20
Number of structures
18
16
14
12
10
8
6
4
2
0
1986
1988
1990
1992
1994
1996
Year
1998
2000
2002
2004
CRYSTALLIZATION OF MEMBRANE PROTEINS
CURRENT OPINION IN STRUCTURAL BIOLOGY, 7, 697-701 (1997).
Crystallization of membrane proteins
Christian Ostermeier and Hartmut Michel
“------ 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.-------”
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 CRYSTALLIZATION
EHUD M. LANDAU AND JŰRG P. ROSENBUSCH
Proc. Natl. Acad. Sci. USA
Vol. 93, pp. 14532–14535, December 1996
Lipidic cubic phases: A novel concept for the crystallization of
membrane proteins
“---- 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 diffracted to 3.7 Å resolution (NOW
TO 1.6 )”
(halorhodopsin and sensory rhodopsin II also crystallized in this
way.)
MEMBRANE PROTEIN STRUCTURES
PNAS 99, 11055 (2002)
Interactions between lipids and bacterial reaction centers determined by protein
crystallography Camara-Artigas, Brune, and Allen Department of Chemistry and
Biochemistry and Center for the Study of Early Events in Photosynthesis, Arizona
State University,
“Three lipid molecules that lie on the surface of the protein are resolved
in the electron density maps. ---cardiolipin ---phosphatidylcholine ---glucosylgalactosyl diacylglycerol. ---- lipids are located in the
hydrophobic region of the protein surface and interact predominately
with hydrophobic amino acids, in particular aromatic residues. Although
the cardiolipin is over 15 Å from the cofactors, the other two lipids are in
close contact with the cofactors and may contribute to the difference in
energetics for the two branches of cofactors that is primarily responsible
for the asymmetry of electron transfer. The glycolipid is 3.5 Å from the
active bacteriochlorophyll monomer and shields this cofactor from the
solvent in contrast to a much greater exposed surface evident for the
inactive bacteriochlorophyll monomer. The phosphate atom of
phosphatidylcholine is 6.5 Å from the inactive bacteriopheophytin, and
the associated electrostatic interactions may contribute to electron
transfer rates involving this cofactor. Overall, the lipids span a distance
of 30 Å, which is consistent with a bilayer-like arrangement suggesting
the presence of an ‘‘inner shell’’ of lipids around membrane proteins that
is critical for membrane function.”
Camara-Artigas et. al
glycolipid
PC
cardiolipin
glycolipid
cardiolipin
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 online textbook].
 membrane protein environment is complex; it involves
the aqueous region outside the membrane, electrical
charges at the membrane surface, and the hydrophobic
interior of the membrane. The steep dielectric gradient
makes it highly unfavorable to bury a charge (20
kcal/mole) or have an unsatisfied H-bond (5 kcal/mole);
this 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 specific protein conformational states.
 Hydrocarbon chain packing may also stabilize specific
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 interior packing as soluble proteins;
helices tilted 20 to facilitate packing between side
chains; H-bonds between helices are rare and salt bridges
rarely found. Because of helix dipoles, antiparallel
arrangement of transmembrane helices preferred. Trp and
tyr mainly present at membrane-water interfaces; act as
"anchors".
SOME EXAMPLES OF MEMBRANE
PROTEIN STRUCTURES
WILL FOCUS MAINLY ON STRUCTURES BUT WILL
ALSO BRIEFLY DESCRIBE FUNCTIONAL PROPERTIES
IN SOME CASES
cyt c binding site
Cu A
heme a
Cu B
heme a3
13 subunits
Cu A
Mg
Cu B
heme a
heme a3
distances:
Cu A
Fe a : 19Å
Cu A
Fe a3 : 22Å
Fe a
Fe a3 : 14Å
CCO COFACTORS
Mills & Ferguson-Miller, BBA 1555, 96-100 (2002)
TWO ROLES:
1- GENERATION OF PMF
2- REDUCTION OF OXYGEN
TO WATER
PROTON TRANSPORT PATHWAYS IN CCO
Zhang et al.,
Nature 392, 677
(1998)
Cytochrome
bc1
11 subunits
dimer
Rieske protein
2Fe-2S
FeS
Two
conformations
observed
Positions of
redox centers
in two
conformations
of Rieske
protein
Lange & Hunte
PNAS, 99, 2800
(2002)
Cytochrome bc1cytochrome c
complex
KcsA Potassium Channel From Streptomyces lividans. 1BL8
tetramer of
identical
subunits
BACTERIORHODPSIN: a light-driven proton pump
R. viridis
Deisenhofer,
Michel and
Huber
Light-harvesting pigment
from photosynthetic
bacteria
McLuskey et al.
Biochemistry 40,
8783 (2001)
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.
PROSTAGLANDIN H2 SYNTHASE
Prostaglandin H2 synthase. 1PRH
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.
Maltoporin Trimer From Salmonella typhimurium. 2MPR
PORIN FROM RHODOBACTER CAPSULATUS
Weiss & Schulz,
J. Mol. Biol. 227,
493 (1992)
Alpha-hemolysin. 7AHL
From Staph.
aureus;
heptameric
pore-forming
protein
Secreted as
33 kD soluble
protein;
aggregates
and inserts
into
membrane.
Pore has
hydrophobic
exterior and
hydrophilic
interior.
Song et
al. Science,
274, 1859
(1996)
SEC Y – PROTEIN TRANSLOCATING COMPLEX
van den
Berg et al.
Nature 427,
36 (2004)
heterotrimer
channel forms
a passive
conduit for
extended
polypeptide
chain
ELECTRON TRANSFER IN OXYGENIC
PHOTOSYNTHESIS
two light reactions bridge energy gap between water and NADPH
Khlbrandt,
Nature 426,
399 (2003).
x-ray structures
for PSI and PSII
have been
determined
cyclic ET
complexes from algae
and cyanobacteria have
essentially the same
structures, despite large
evolutionary separation
cofactors (per monomer):
4 hemes
1 2Fe-2S cluster
1 chlorophyll a
1 β-carotene
1 plastoquinone
no evidence
from structures
for movement
of Rieske protein;
however,
presumed to
occur because of
long distance
between FeS and
heme f (29 Å);
hinge region
contains many
glycine residues
Kurisu et al. Science 302, 1009 (2003)
13 transmembrane
helices per
monomer
central lipidfilled cavity
subunits:
cyt b6
cyt f
Rieske ISP
subunit IV
PetG
PetL
PetM
PetN
TDS: quinone analog
inhibitor
Mol. wt. =
217 kD
space-filling view of cyt b6f
TDS bound on
p-side and PQ
on s-side;
consistent with
transfer of PQ
between
monomers
central cavity
( 2 DOPC
molecules
bound)
note bound plastoquinone
location of chlorophyll a (function unknown);
no clear axial ligand can be seen (may be a
water molecule bridging to a peptide carbonyl);
edge exposed to lipid phase
location of β-carotene: near center of
transmembrane region; too far from chl a
to quench triplet state (function unknown)
heme X may
function in cyclic
electron transfer
(does not occur
in cyt bc1); could
mediate flow of
electrons between
PQ and ferredoxin
Environment of heme X (heme ci): covalently
bound by one thioether linkage; has no axial amino
acid ligand (1 water bound); located between
heme bn and central cavity; note location of PQ
ION TRANSPORT: POTASSIUM CHANNELS
KcsA from Streptomyces lividans: a diffusive K+ channel
Doyle et al. Science 280,
69 (1998)
main questions:
1- selectivity; K+ (radius
1.33 Å) >104 Na+ (radius
0.95 Å)
2- high throughput;
approaches diffusion limit
( 108 per sec)
pore helix
(points toward
center of cavity)
Streptococcus
lividans KcsA
channel; tetramer
of 4 identical
subunits
3.2 Å resolution
each subunit has
2 transmembrane
helices; one faces
the central pore and
the other the lipid
phase; sequence
conservation among
K+ channels
strongest for pore
region and inner
helix
distribution of aromatic residues; form 2 layers near
membrane-water interfaces; help to position channel in
membrane
red: negative charge
blue: positive charge
white: neutral
yellow: hydrophobic
green spheres: K+ ions
note negative
potential near
entrance and
exit and non-polar
region in between;
this distribution of
charge facilitates
high throughput
cutaway view showing solvent-accessible surface
and distribution of electrical charge
total length 45 Å;
selectivity filter is
12 Å long (minimizes
distance over which
K+ interacts strongly
with channel)
diameter of
selectivity filter
too narrow to
accommodate
hydrated K+ ion;
thus waters must
be stripped off for
ion to enter
central cavity
and following
region large
enough to be
filled with water
view showing pore dimensions
conduction occurs when one ion enters from one side and a second exits
from the other side; ions in selectivity filter repel each other and force one to
move into central cavity; direction of flow determined by concentration
gradient
pore helices
K+ in central cavity is fully
hydrated [cf. Zhou et al. Nature 414,
43 (2001); also contains a
discussion of the entry of K+ into the
channel and of effect of K+
concentration on channel structure]
energy barrier to movement of ions through membrane is highest at the center;
having water molecules in center and oriented helix dipoles compensates for
this; hydrophobic lining of channel prevents ions from sticking to surface; having
two ions in selectivity filter causes structure change that facilitates movement
selectivity filter:
composed of four evenly
spaced layers of
carbonyl oxygen
atoms and one layer of
threonine hydroxyl
oxygens
Gly residues
allow all carbonyl
oxygens to
point in same
direction
side view of K+ ions in selectivity filter; note coordination
by carbonyl oxygens that closely mimic, and compensate for loss
of, waters of hydration
view from top through selectivity filter; note close fit and coordination of K+
by carbonyl oxygens
view down through selectivity filter showing network
of aromatic residues surrounding pore; acts to hold the
filter open to prevent accommodation of smaller Na+
KvAP from Aeropyrum pernix: a voltage-gated K+ channel
Jiang et al. Nature 423,
33 (2003)
structure of KvaP – central pore is
essentially identical to that of KscA;
structures deviate beginning at the
intracellular membrane leaflet
Fab fragments
gly gating
hinges
KvaP – blue
KscA - green
crystallized as antibody
complex
voltage sensor paddles
(helices 3b and 4) –
located on perimeter of
pore and connected to
main part of channel by
3a helix and sharp turn at
helix 5; should allow free
movement relative to
channel
close-up view of isolated voltage
sensor paddle – 4 arg residues
(117, 120, 123, 126) shown by
mutation to be essential for gating
(rest of sequence is hydrophobic);
R133 forms salt bridge with D62,
joining S4 and S2
comparison of structure of
isolated voltage paddle (b)
with intact channel (a);
provides evidence that paddle
is highly flexible and is loosely
packed against the pore;
suggests that it acts as a
separate mobile domain
(consistent with functional
voltage-gated channel being
produced by splicing a voltage
paddle with the KcsA channel)
voltage paddle
model for voltage-gating: + charges on paddle (arg residues) are
carried through the membrane by passage of action potential and
act to open pore; consistent with experiments in which tethered
biotin is accessible to avidin from the intracellular side when the
channel is closed and accessible from the extracellular side when
the channel is open; mechanism implies that hydrophobic and
electrostatic forces can balance each other to allow charges to be
pulled through the membrane interior. Model still controversial.
MscL from Mycobacterium tuberculosis: a mechanically
gated K+ channel
structure determination: Chang et al. Science 282, 2220 (1998) – gated open
by increase in lateral tension applied to the bilayer; non-selective; thought to
protect against osmotic stress; crystal structure corresponds to closed state
tetrameric
comparison of channel structures
(note cytoplasmic domain of 5-helix
bundle in MscL extending ~35 Å
from membrane)
pentameric; threaded
across membrane in
opposite manner to
KcsA (N-terminal helix
is inner); outer helices
contact inner helix of
adjacent subunit
gating mechanism: structure of open state still not known, but
high non-selective conductance consistent with absence of
selectivity filter and suggests water-filled open pore diameter
of ~ 40 Å.
working model: lateral pressure in membrane clamps the
channel in the closed state; rearrangements in the lipids packed
around the channel in response to membrane stretching
reduces the pressure and allows the channel to open via
changes in helix tilt; role of extramembrane domain is uncertain
(much of it can be removed without influencing function).
(cf. Perozo and Rees, Curr. Opin. Struct. Biol. 13, 432 (2003) for
additional discussion)
PROTON PUMPING BY BACTERIORHODOPSIN
PROTON TRANSPORT IN BACTERIORHODOPSIN
Simplest known example of a transmembrane ion pump; proton pumped
from cytoplasm to outside; gradient utilized for ATP synthesis. Elucidation of
mechanism of action resulted mainly from combination of crystallography
and time-resolved spectroscopy (UV/vis; FTIR; resonance Raman; NMR).
structure: Luecke et al.
J. Mol. Biol. 291, 899
(1999).
retinal chromophore
attached to lys residue
via protonated Schiff
base
MW ~ 24 kD
7 transmembrane
helices
N-terminus
C-terminus
H
H
photoreaction causes isomerization
of 13,14 double bond from trans to cis
conformation
BACTERIORHODOPSIN PHOTOCYCLE
From Neutze et al.
BBA 1565, 144
(2002)
photoisomerization
re-isomerization
from
H+
cytoplasm
internal proton
transfer
to extracellular H+
medium
proton release and proton uptake separated in time and space; protein
relaxation much slower than photoisomerization
from Mathies et al.
Ann. Rev. Biophys.
Biophys. Chem. 20,
491 (1991).
Mechanism of photoisomerization
From Neutze et al.
BBA 1565, 144 (2002)
SEQUENCE OF EVENTS
IN PROTON TRANSPORT
PATHWAY IN
BACTERIORHODOPSIN
transmembrane helices
enclose a cavity that spans
the membrane; retinal
divides the cavity into two
sections:
1-extracellular (hydrophilic,
wide); has H-bonded
network of 4 residues
(arg82, tyr57, glu194,
glu204), and at least 6
bound water molecules.
2-cytoplasmic hydrophobic,
narrow); contains only one
residue involved in proton
transport (asp96; has
unusually high pK and is
protonated in the dark),
and fewer bound waters;
has to undergo
conformational changes
during photocycle that
allow water to enter.
From Lanyi, on-line
textbook
active site can be
thought of as
consisting of a water
molecule (W402)
coordinated by the
protonated retinal
Schiff base, which is
salt-linked to two
anionic asp residues,
asp85 and asp212.
This charge pairing,
plus additional Hbonds, stabilizes the
buried charges.
Schiff base is
deprotonated during
the photocycle.
DARK ADAPTED STATE
buried charges
unusual for a
membrane
protein
From Neutze et al.
BBA 1565, 144 (2002)
note H-bond
between D96
and T46; raises
pKa of D96,
inhibiting proton
transfer to
Schiff base
view of cytoplasmic channel in dark-adapted
state showing distorted α-helix G (so-called π-bulge)
and single bound water
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 (dark) reactions of the photocycle.
Proton is transferred from Schiff base to asp85 within about 50
s; helped by a small movement of helix C which brings them closer
together. Proton may be derived directly 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 Hbonds form to carboxyl group), or indirectly from the bound water
molecule, generating hydroxyl ion which removes proton from retinal.
Protonation states of asp85, glu204 and glu194 are linked.
Transfer of proton to asp85 moves it away from retinal and allows
movement of arg82 towards bottom of channel. This causes pK of
glu204 to decrease; glu204 transfers proton to glu194, which causes
proton release at the extracellular surface.
From Luecke et al.
Science 286, 255 (1999)
purple – dark
yellow – M state
isomerization of
retinal reverses
direction of
Schiff base and
causes steric
clash with W402
note aromatic
residues
flanking retinal;
these act to
immobilize
polyene chain
and move in
response to
photoisomerization;
results in movement
of helix F
From Luecke et al.
Science 286, 255 (1999)
dark state
STRUCTURAL CHANGES
IN EXTRACELLULAR
CHANNEL UPON
FORMATION OF M
STATE
loss of H-bond
loss of W402
M state
movement of R82,
E194 and E204
Reprotonation of Schiff base from cytoplasm requires that pK of asp96
be lowered and proton pathway created (probably via bound water).
Protein conformation change in M intermediate is caused by retinal
isomerization; the 13-methyl group pushes on trp182, moving helix F.
This opens cytoplasmic channel (aided by a local unwinding of helix G)
and allows water to enter, which causes pK of asp96 to decrease;
results in proton transfer to Schiff base.
DARK RE-ISOMERIZATION OF RETINAL
Causes reversal of protein conformational change by removing steric
clash with trp182. Restores the high pK of asp96, leading to
reprotonation from cytoplasm.
Final proton transfer occurs from asp85 to glu204 (via arg82 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.
From Neutze et al.
BBA 1565, 144 (2002)
structure of early M intermediate in mutant BR
showing increased water content near asp96
and increased accessibility to Schiff base
structural relaxation
recovers original
conformation
outward movement
of helix F opens
cytoplasmic
channel, enabling
water to enter,
proton transfer
to Schiff base, and
reprotonation of
asp96
local bend of
helix C enables
proton transfer to
asp85
protein conformation changes occurring during photocycle
From Lanyi and Schobert, Biochemistry
43, 3-8 (2004)
Strained conformation in K stores
free energy; in subsequent steps
retinal relaxes; coupled to protein
conformational changes.
RETINAL MOVEMENT DURING
FIRST HALF OF PHOTOCYCLE
Retinal distorted due to counter-rotations
of C14-C15 and C5-N bonds; H-bond to
water broken
Schiff base N-H realigns with water
Schiff base proton transferred to Asp-85
M1 to M2 is the reprotonation switch
Schiff base N fully rotated to cytoplasmic
side
Retinal fully bent
MOVEMENT OF ARG-82 TOWARDS EXTRACELLULAR SURFACE
CAUSING PROTON RELEASE
FORMATION OF WATER CHAIN IN CYTOPLASMIC REGION
DURING SECOND HALF OF THE PHOTOCYCLE
13-methyl group pushes against
indole ring of Trp-182 in M2; Lys216 side chain twists. Leads to
repacking of side chains between
helices F and G that results in
outward tilt of helix F, inward tilt of
helix G and formation of a cluster of
water molecules between Asp-96
and Schiff base N.
pKa of Asp-96 is lowered causing
reprotonation of Schiff base.