Biochem 503
Membrane Structure & Properties
(Michael Wiener, mwiener@virginia.edu, 3-2731, Snyder 360)
• What is the major structural component of biological membranes?
• Why do bilayers form?
• What is the structure of a lipid bilayer?
• What properties are conferred upon membranes by this structure?
References:
Books
* Robert M. Gennis, Biomembranes, Springer-Verlag, 1989.
Charles Tanford, The Hydrophobic Effect: Formation of Micelles and Biological Membranes,
John Wiley & Sons, 1980.
Donald M. Small, The Physical Chemistry of Lipids (Handbook of Lipid Research Vol. 4),
Plenum Press, 1986.
Derek Marsh,, CRC Handbook of Lipid
p Bilayers,
y , CRC Press,, 1990.
Papers
R. McElhaney, "The influence of membrane lipid composition and physical properties of
membrane structure and function in Acholeplasma laidlawii," Crit. Rev. in Microbiol. 17:1-32
(1989).
S. McLaughlin, "Electrostatic Potentials at Membrane-Solution Interfaces," Cur. Topics in
Membranes
emb anes and Transport,
anspo t, 9:7
9:71-144 ((1977).
977).
B. Honig, W.L. Hubbell & R.F. Flewelling, "Electrostatic Interactions in Membranes and
Proteins," Ann. Rev. Biophys. Biophys. Chem., 15:163-193 (1986).
W.C. Wimley & S.H. White, "Experimentally determined hydrophobicity scale for proteins at
membrane interfaces," Nature Struct. Biol. 3:842-848 (1996). {Also refer to comments by Deber
& Goto on pp. 815-818}.
Wiener, M.C. and White, S.H. (1992). Structure of a fluid dioleoylphosphatidylcholine bilayer
determined by joint refinement of x-ray and neutron diffraction data. III. The complete structure.
Biophys. J., 61:434-447.
Journals
Biochemistry, Biophysical Journal
Membrane Structure
Lipids are amphipathic molecules, consisting of hydrophobic and hydrophilic portions. Nonpolar
hydrocarbon chain(s) are attached to a polar headgroup via a backbone structure, typically
glycerol.
glycerol
The most common class of lipids are the phospholipids, with different headgroups shown.
What happens when an amphipathic molecule such as a lipid is dispersed or dissolved in an
aqueous solution?
The energetics involved in this process drive the self-assembly of amphiphiles into structures
such as bilayers and micelles
micelles. The behavior of amphiphiles in aqueous solution is often
described by the term "Hydrophobic Effect."
What is the Hydrophobic Effect?
Gibbs free energy
Gibbs free energy: G = H - TS
H: enthalpy (chemical bonds, steric, electrostatic)
S: entropy = k lnQ (number of configurations)
ΔG = ΔH - TΔS
The properties of water around solutes
Liquids do not possess long-range order, i.e., there is not a regular network of interactions
extending over many molecules in the bulk phase. However, liquids are not random collections
of particles. A well-defined nearest-neighbor separation can be obtained from x-ray scattering
experiments. In liquid water, this distance is ~2.8Å. A water model that can explain all of the
properties
ti off liquid
li id water
t has
h nott yett been
b
developed.
d l d However,
H
in
i mostt models,
d l many or all
ll
water molecules form hydrogen bonds to neighboring water molecules, but the bonds can be bent
or stretched to produce an irregular network. When a polar or charged molecule is dissolved in
water, ordered clusters of water form around the solute. This can be seen in crystal structures of
clathrate compounds.
The number of conformations of the water molecules around the solute is decreased relative to
the conformations of the molecules in the bulk. Thus the (conformational) entropy of the system
decreases with the presence of the solute. For polar or charged molecules, this decrease in
entropy (which increases ΔG) is offset by the decrease in enthalpy (which decreases ΔG) of the
system.
For a pure hydrophobic compound (e.g., alkane, olive oil), the water will be ordered around this
solute but there is no corresponding enthalpy to make ΔG favorable. Water will exclude these
compounds from solution. This is reflected in the very low solubilities of hydrocarbons in water.
In general, purely hydrophobic compounds are nearly immiscible in water.
Since the unfavorable conformational entropy arises from ordering of water molecules around a
nonpolar
l surface,
f
increasing
i
i the
h surface
f
area off this
hi nonpolar
l region
i makes
k it
i more energetically
i ll
unfavorable.
What about amphipathic molecules, which have both polar and nonpolar parts? There will be a
balance between the favorable ΔG of solvating the polar/charged headgroup, and the unfavorable
ΔG to accomodate the hydrocarbon region. One prediction would be that amphipathic molecules
would have a low solubility, a bit higher than pure hydrophobics, and be nearly immiscible. But,
fortunately for life as we know it, the amphipathic chemical nature of these molecules leads to a
spontaneous self
self-organization
organization into structures that minimize the free energy of the system.
system
Specifically, structures form which sequester the hydrophobic parts of the molecule away from
water and solvate the polar/charged parts of the molecule.
The concentration dependence of amphiphile self-assembly into micelles, bilayers, etc. is
characterized by the critical micelle concentration (CMC).
What is the structure of lipid molecules in a bilayer?
Crystal structures of lipids show the conformations of the individual molecules.
.
Crystal structures of lipids also pack as bilayers. However, lipid bilayers and biological
membranes
b
are referred
f
d to as li
liquid-crystalline
id
lli systems. Wh
What is
i missing
i i from
f
these
h
structurall
images?
Trans-gauche isomerization of acyl chains.
Introduction of kinks into acyl chains in pure hydrocarbons is associated with the first-order
phase transition between solid and liquid phases. In lipid bilayers, there are phases that have alltrans acyl chains like solid alkanes, but these phases are generally not present in biological
membranes in vivo. The acyl chains are melted, and the bilayer interior is more like a liquid. Xray scattering of liquid alkanes or of the melted acyl chains of a bilayer are qualitatively similar
q
water; there is an average
g distance but not an ordered lattice.
to liquid
The fluid state of the membrane is essential for cell viability. Some bacteria are fatty-acid
auxotrophs, and need to be supplemented with fatty acids in order to grow. These organisms can
have their membrane composition manipulated by addition of different fatty acids. It has been
shown that the growth temperature of these bacteria is always at or above the melting
temperature of the lipids that comprise their membranes.
NMR spectroscopy can be used to probe the conformation of the acyl chains in a bilayer. Lipids
with the hydrogens of the acyl chains replaced by deuterons are utilized. The C-D bonds in each
methylene
th l
group define
d fi a plane,
l
in
i which
hi h a vector
t rCD lies.
li
The
Th vector
t rCD makes
k an angle
l θ
with respect to the molecule normal rmol.
The average value of θ can be used to define an order parameter SCD:
SCD = 1/2 (3<cos2θ> - 1),
where <cos2θ> is a time average. The molecular order parameter Smol is often used:
Smol = -2SCD
What does the structure of this liquid-crystalline bilayer look like? The bilayer is like a onedimensional crystal along the bilayer normal, with liquid properties in the plane of the
membrane. By carrying out x-ray and neutron diffraction of lipid bilayers and developing
appropriate theories and models, a structure of a liquid crystalline bilayer can be determined.
The different pieces of the lipid have distributions of different widths and positions. These
distributions are the long-time average and are wider than the 'steric' size because of thermal
motion associated with the liquid crystalline state.
Membrane Properties
Lipid
p bilayers
y are veryy ggood electrical insulators
A typical membrane potential is about 100 mV, and the thickness of the bilayer is about 50Å.
The dielectric breakdown of insulators is given in terms of V/cm.
100 mV
= 200,000 V/cm
50 Å
Ceramic insulators have a breakdown at ~ 150,000 V/cm.
Lateral diffusion in membranes
A diffusion constant D (cm2/sec) can be determined by fluorescence methods. Translational
diffusion constants range from 10-7 to 10-12 cm2/sec. For lipids in fluid membranes, D is
typically 10-8 cm2/sec, which corresponds to a movement of about 1 μm in a second. The area of
a single lipid molecule is about 50 Å2, so this value of 10-8 cm2/sec is an area of about 2,000,000
lipid molecules.
Impermeability of membranes to inorganic ions
Membranes are almost completely impermeable to Na+, K+, Ca++, Cl-, etc. The energy barrier
arises from the very unfavorable electrostatic energy to bring an ion across the nonpolar bilayer
interior. Water is a high-dielectric medium with a dielectric constant of ε ≈ 80, while the bilayer
interior has a value of ε ≈2-3.
≈2 3
The Born equation describes the free energy of transfer to move a charged object of radius r from
a region of dielectric constant ε2 to a a region of dielectric constant ε1:
WB =
q 2 ⎜⎛ 1
1⎞
− ⎟
2r ⎝ ε 1 ε 2 ⎠
This is about 81 Z2/r Kcal/mole. This is the main contribution to the energy barrier. Other lesser
effects arise from image energy, dipole energy, and neutral energy.
Biochem 503
Membrane Protein Structure
(Michael Wiener, mwiener@virginia.edu, 3-2731, Snyder 360)
• 2D and 3D crystallization of membrane proteins
• Structures of membrane proteins obtained by electron and x-ray
x ray crystallography
• General features of membrane protein structure
References:
Books
Robert M. Gennis, Biomembranes, Springer-Verlag, 1989.
Stephen H. White (Ed.), Membrane Protein Structure, Oxford, 1994.
Reviews
Rees, D.C., Komiya, H., Yeates, T.O., Allen, J.P., and Feher, G. “The bacterial photosynthetic
reaction center as a model for membrane proteins,” Ann. Rev. Biochem. 58:607-633 (1989).
Stowell, M.H.B. and Rees, D.C. “Structure and stability of membrane proteins,” Adv. Prot.
Chem 46:279
Chem.
46:279-311
311 (1995).
(1995)
Papers
Wallin, E. & Von Heijne, G. “Genome-wide analysis of integral membrane proteins from
eubacterial, archaean, and eucaryotic organisms,” Protein Sci. 7:1029-1038 (1998).
Baldwin, J.M., Henderson, R., Beckman, E. & Zemlin, F. "Images of purple membrane at 2.8Å
resolution obtained by cryo-electron microscopy," J. Mol. Biol. 202:585-591 (1988).
U i N
Unwin,
N. "P
"Projection
j ti structure
t t
off the
th nicotinic
i ti i acetylcholine
t l h li receptor:
t distinct
di ti t conformations
f
ti
off
the a subunits," J. Mol. Biol. 257:586-596 (1996).
Jap, B.K., Downing, K.H. & Walian, P.J. "Structure of PhoE porin in projection at 3.5Å
resolution," J. Struct. Biol. 103:57-63 (1990).
Mitra, A.K., van Hoek, A.N., Wiener, M.C., Verkman, A.S. & Yeager, M. "The CHIP28 water
channel visualized in ice by electron crystallography," Nature Struct. Biol. 2:726-729 (1995).
S h l G
Schertler,
G.F.X.,
F X Vill
Villa, C
C. & H
Henderson,
d
R
R. "P
"Projection
j i structure off rhodopsin,"
h d i " Nature
N
362:770-772 (1993).
De Rosier, D.J. & Klug, A. "Reconstruction of three dimensional structures from electron
micrographs," Nature 217:130-134 (1968).
Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckman, E. & Downing, K.H. "Model
for the structure of bacteriorhodopsin based on high-resolution electron cryomicroscopy," J. Mol.
Bi l 213:899-929
Biol.
213 899 929 (1990).
(1990)
Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. & Rees, D. "Structure of the reaction center
from Rhodopseudomonas sphaeroides R-26: the protein subunits," Proc. Natl. Acad. Sci. USA
84:6162-6166 (1987).
Weiss, M.S. & Schulz, G.E. "Structure of porin refined at 1.8Å resolution," J. Mol. Biol.
227:493-509 (1992).
Song, L., Hobaugh, M., Shustak, C., Cheley, S., Bayley, H. & Gouaux, J.E. "Structure of
staphylococcal α−hemolysin, a heptameric transmembrane pore," Science 274:1859-1866 (1996).
{Also refer to comments by D.M. Engelman on pp. 1850-1851}
Pebay-Peyroula, E., Rummel, G., Rosenbusch, J.P. & Landau, E.M. "X-ray structure of
bacteriorhodopsin at 2.5Å from microcrystals grown in lipidic cubic phases," Science 277:16761681 (1997) {Also refer to comments by A. S. Moffat on pp. 1607-1608}
Kimura, Y., Vassylyev, D.G., Miyazawa, A., Kidera, A., Matsushima, M., Mitsuoka, K., Murata,
K., Hirai, T. & Fujiyoshi, Y. "Surface of bacteriorhodopsin revealed by high-resolution electron
crystallography " Nature389:206-211(1997).
crystallography,
Nature389:206 211(1997)
What
h do
d we mean by
b structure??
There are many experimental and theoretical approaches that yield "structural" results. For
example, the primary structure is encoded in the protein sequence. Functional studies can
pinpoint specific amino acids which are essential for protein activity. Optical spectroscopy
(circular dichroism & infrared) provides information on secondary structure, and can provide
reasonable estimates of the amounts of α−helices,
α helices β−sheet
β sheet and random coil in a protein.
protein
Fluorescence spectroscopy can report on the chemical environment of a fluorophore (e.g.,
aromatic side-chains), with fluorescence intensity, wavelength & 'quenchability' indicating
whether the protein is in an aqueous or membrane environment. With appropriate experiments,
fluorescence can also determine distances between pairs of amino acids. These types of results
can also be obtained, often more effectively, with EPR spectroscopy. For soluble proteins, NMR
can be used to obtain many pairs or distances, which can be used to determine a protein structure.
{Why do you think that this might not work so well with membrane proteins?} For ion channels,
electrical measurements combined with mutagenesis & pharmacology can map out structural
aspects of channels. Other biochemical & biophysical methods (cross-linking,
ultracentrifugation, mass spectroscopy, ...) provide different types of information that can be said
p
structure.
to have somethingg to do with protein
For the purposes of today's lecture, structure will refer to information about the entire protein (or
at least its membrane-bound form) obtained to moderate to high resolution. Electron
crystallography is not often able to yield complete three-dimensional structures, but is a very
powerful method for analysis of membrane proteins. X-ray crystallography has been the most
powerful method to date for the determination of three-dimensional structures of integral
membrane
b
proteins
i to atomic
i resolution.
l i
Several
S
l other
h experimental
i
l techniques
h i
may also
l be
b
covered today.
Membrane proteins are ubiquitous, and carry out many biological/biochemical functions. These
functions include: channels, transporters, receptors and enzymes.
Genomic analysis indicates that ~20-30% of all proteins are membrane proteins.
Yet, there are relatively few structures to date.
Conformation and sequence of the polypeptide chain within the membrane
In solution, the amide backbone of a protein can hydrogen-bond with other amino acids or with
solvent molecules (i.e.,
(i e water).
water) In the membrane interior there is nothing to H
H-bond
bond with except
the polypeptide chain itself. The energetic cost of burying an unsatisfied H-bond is high enough
to exclude or greatly reduce the possibility of "random coil" structures within the bilayer.
Therefore, the elements of the protein in the membrane and in contact with lipid are likely to be
regular secondary structure elements (α−helix, β−sheet) in order to form intramolecular
hydrogen bonds.
It is very unfavorable to partition a charge into the membrane interior. Therefore, one can expect
that for the portion of the polypeptide that faces lipid, there should be a lack of charged amino
acids. Polar amino acids that, in solution, are able to h-bond with water will not be able to form
h-bonds with the aliphatic lipid chains. Therefore, one might also expect that there will be few
polar residues in the parts of the polypeptide that face the lipid.
Electron Crystallography
Under favorable conditions, two-dimensional crystals of membrane proteins can be grown.
Typically, the membrane protein is extracted from the native membrane with detergent and
purified with micelles of detergent replacing the lipid of the native membrane. Lipids are added
to this solution
solution, and the mixture is dialyzed to slowly remove detergent.
detergent Small ordered patches
(two-dimensional arrays) of membrane embedded protein can grow, and can be examined by
imaging and diffraction of electron beams. These samples are only one membrane in thickness a monolayer of protein and a bilayer of lipid! Electrons scatter from the electrons in the sample
so the resultant image is a map of the electron density of the object.
Images --> direct experimental determination of phases
Diffraction --> direct experimental determination of amplitudes
Unlike x-ray crystallography, there is NO phase problem!
Orienting the sample with the membrane surface perpendicular to the electron beam yields a
projection structure. This two-dimensional structure can be very informative about the structure
of the protein.
The 6Å projection of human AQP-1, determined by Mitra et al. The dotted line encircles an AQP-1 monomer, and
the contours labelled 1-6 are consistent with a-helices in projection. The scale bar is 10Å. The two-dimensional
crystal is in plane group p4g, with a=b=100Å. There are two tetramers in the unit cell.
At low resolution, interpretation of the electron density can be quite difficult.
It is possible, by tilting the sample, to determine the three-dimensional structure of the protein
from a two-dimensional crystal. Several three-dimensional structures have been determined in
this
hi fashion,
f hi although
l h h there
h is
i a "missing
" i i cone problem"
bl " due
d to the
h geometry off the
h data
d
collection.
Using this method, several membrane protein structures have been determined by electron
crystallography. An example is the Kimura et al. bacteriorhodopsin paper. However, the
resolution is much lower than that obtainable (in principle) from x-ray crystallography of threedimensional crystals, and the electron-crystallographic structures have anisotropic resolution
because of the 'missing cone' problem. It is likely that many membrane proteins will be (and
already are being) studied with both methods.
Three-dimensional membrane protein crystals are ordered arrangements of a protein-detergent
complexes (PDCs). With nearly no exception, the packing of PDCs in the crystal lattice does
not recapitulate the membrane structure, i.e., membrane protein crystals are of type II as indicated
above.
b
IIn contrast,
t t two-dimensional
t
di
i l membrane
b
protein
t i crystals
t l usedd for
f electron
l t
crystallography
t ll
h
are one layer thick and can be thought of as an ordered two-dimensional aggregate in the plane of
the membrane. Two-dimensional crystals are formed by a variety of ways; most commonly,
detergent-solubilized protein is mixed with lipids and this mixture is dialyzed to allow the
detergent to diffuse away. Membrane sheets form, and the high density of protein in the
membrane leads (when successful!) to the formation of ordered regions.
Examining Membrane Protein Structures
Bacteriorhodopsin
Bacteriorhodopsin has arguably been the most extensively studied integral membrane protein. It
is from a halophilic archaebacterium, Halobacterium halobium, and occurs naturally in localized
high-density patches called purple membrane. It has a molecular weight of about 27,000 and has
a single covalently bound prosthetic group, retinal. It acts as a photochemical protin pump to
create a proton electrochemical potential difference across the membrane. The existing structure
h been
has
b
determined
d
i d by
b electron
l
crystallography,
ll
h andd recently
l by
b x-ray crystallography
ll
h andd 'high'hi h
resolution' electron crystallography.
There are seven α−helices, each of 20-25 predominantly hydrophobic residues, which are able to
span the membrane
membrane. The helices are arranged in two layers; each layer has three or four helices
helices.
The α−helices are arranged in a simple up-and-down pattern with helical regions adjacent in
sequence also being adjacent to each other in structure. The helical packing is similar to that
observed in soluble helical bundle proteins or coiled-coil proteins. This "knobs-into-holes"
helical packing with a crossing angle of about 23° is a packing motif involved in the formation
of compact domains from α−helical secondary structural elements. Most of the charged or polar
residues are in the extracellular loops, although there are as many as nine ionizable groups within
the membrane. These are likely to be involved in salt bridge (ion pair) formation with other
sidechains
id h i or the
h retinal.
i l Many
M
off the
h aromatic
i residues,
id
Trp
T andd Tyr,
T are near the
h membrane
b
surface (!).
The photosynthetic reaction center (PSRC) of Rhodobacter sphaeroides
Photosynthetic reaction centers are protein-pigment complexes which carry out the primary
charge separation in photosynthetic membranes. The protein consists of three subunits (H, L and
M) and more than ten prosthetic groups. The molecular weight is about 150,000. The structure
has been determined by x-ray crystallography
crystallography. The transmembrane regions are α
α−helical
helical, and
nearly all of the residues in these helices are nonpolar. Most of the helices are antiparallel. Most
of the helices are tilted 25° or less with respect to the bilayer normal, but there are a few helices
that tilt by almost 40°.
1.
Another class of integral membrane protein structures are those whose membrane-spanning
regions are comprised of β−sheets. All of the proteins found in the outer membranes of bacteria,
to date, are these β−barrel proteins. Changing the number of strands changes the diameter of the
barrel Figs
barrel.
Figs. a and b are porins,
porins each comprised of a single polypeptide chain.
chain Fig.
Fig c depicts
α−hemolysin, a toxin that is secreted by bactera and assembles to subsequently form a large and
lethal pore in the membranes of ‘attacked’ cells. This structure is a heptamer (n=7) of
interweaving monomers.
A striking feature is the position of aromatic amino acid residues in the structures. Rather than
partitioning into the membrane interior, as one might have expected from the large hydrophobic
surface areas of their side-chains, these residues are located approximately at the membrane
surface. More specifically, comparison with studies of model peptides in lipid bilayers suggests
that these residues are located in the interfacial region, i.e., the region of a lipid bilayer which
bounds the hydrophobic interior and that is comprised of the headgroups, backbones, water, etc.
This pposition of the aromatics is also observed in α−helical membrane pproteins.
Given the structures of integral membrane proteins determined to date, what structural principles
or observations can be made?
1. Surface areas of membrane proteins are similar to those of soluble proteins. Even though the
membrane environment of the membrane-embedded portion of a membrane protein is very
different from the aqueous environment of soluble proteins, the surface areas of each per
mass are about the same.
2. The interior of the portion of membrane proteins that are embedded in the membrane is about
as nonpolar as the interior of soluble proteins. Of course, the lipid-facing residues of
transmembrane segments are nonpolar, compared to the polar/charged residues of soluble
proteins. Therefore, membrane proteins are NOT soluble proteins turned inside out!
3. The
h packing
ki volumes
l
off buried
b i d residues
id
in
i membrane
b
proteins
i andd soluble
l bl proteins
i are
similar, indicating that similar packing is used in bot types of proteins.
4. As expected, charged side chains are not exposed to the membrane interior. Transmembrane
helices can be slightly tilted, but the general effect is to orient the helix dipoles approximately
normal to the membrane surface.
surface
5. Transmembrane helices pack efficiently by maximizing van der Waals interactions and
minimizing cavities between helices.
6 Aromatic residues (Tyr,
6.
(Tyr Trp,
Trp Phe) appear to play a special role in membrane proteins.
proteins These
groups are localized at the surfaces of the membrane, between the hydrophobic membrane
interior and the polar headgroup/interface region.
7. Only a small number of membrane protein structures have been determined to moderate-high
resolution by electron or xx-ray
ray crystallography. Nearly all eucaryotic membrane proteins
(transporters, receptors, channels) remain unsolved.