Chem*3560
Lecture 24:
Membrane proteins
Biological membranes are rich in protein, and although proteins can't be "seen" in the normal sense (the
smallest visible object is about 250 nm in diameter and proteins are about 5-10 nm), they can made
visible in the light microscope by attaching fluorescent probe molecules such as fluorescein. When
irradiated with blue light, the probe emits yellow green light, which shows the location of the protein (to
within a radius of 250 nm).
This reveals that many membrane proteins are in constant motion as if they are floating on the surface of
a water droplet. They are in fact floating in the fluid lipid bilayer that makes up the membrane, and
motion is diminished at temperatures below the phospholipid transition point.
Two cells are each tagged
on their surface exposed
membrane proteins with a
different color probe
molecule, and then the
cells are fused together.
Within a few minutes, the
proteins intermingle by
diffusion across the membrane surface (Lehninger p.395).
Proteins can also be seen in the electron microscope, but since electron microscopes operate in
ultrahigh vacuum, the sample has to be fixed and preserved, often by coating with a thin film of metal, so
electron microscope images are always static and dead (Lehninger, p.396, Box 12-1).
Proteins location with respect to the bilayer can be examined experimentally in two ways:
Treatment of whole cells with trypsin:
Trypsin is a large soluble protein, unable to enter or cross the
membrane bilayer. Trypsin can only attack polypeptide that is
exposed on the exterior. The relative size of fragments can be
measured by electrophoresis, so that the location of the cuts can
be identified and the portions of the polypeptide exposed outside
the cell can be mapped out.
Permeant and impermeant probes:
Chemically reactive probes can be made in polar and nonpolar
forms. The polar form is unable to cross the membrane ("Imp"
for impermeant in the figure) whereas the nonpolar probe
("Perm") permeates across the membrane and reacts with protein
on both sides.
Proteins that react with the impermeant probe must
be exposed on the outstide.
Proteins that react only with the permeant probe
must be inside the cell.
Membrane proteins can be distinguished from
internal cytoplasmic proteins by lysing the cell. e.g.
by osmotic shock, and washing away the
cytoplasmic contents. The membrane can then be
resealed as an empty membrane "ghost", and the
probe reagents applied to detect which proteins
really are part of membrane structure.
By adjusting conditions for osmotic shock and
resealing, the cell membrane can be resealed inside
out, and tested with probes once more.
This should indicate the opposite orientation.
Integral and peripheral proteins
By adjusting conditions as the the cell cytoplasm is washed away, some proteins were found to stay
with the membrane under mild conditions, but were washed away when more vigorous treatments were
applied, such as washing in high NaCl (which disrupts ionic interactions) or EDTA solutions. EDTA
chelates and removes Ca2+, and Ca2+ is frequently found to act as a bridge between two negative
molecules (Lehninger p. 398-399).
Peripheral membrane proteins are those which are loosely membrane associated, and attach either
to the phospholipid head groups, or to the exposed regions of transmembrane or integral membrane
proteins.
Integral membrane proteins are physically embedded in the bilayer, and are only released by use of
detergents that totally disrupt the bilayer organization. Detergents are micelle forming molecules, and the
integral membrane protein is released, but in a form embedded in a detergent micelle. Integral
membrane proteins have nonpolar amino acids exposed on that part of the surface that contacts the
hydrocarbon core of the bilayer, and this portion ends up in contact with the micelle.
Lipid anchored proteins are not true transmembrane proteins, but more physically attached to the
bilayer than peripheral proteins. These include proteins with an amino acid modified by attaching a fatty
acid or other hydrocarbon chain, e.g. palmitate (16:0) bonded as an ester to Cys or Ser side chains,
or myristate (14:0) as an amide bonded to to the N-terminal amino group.
The attached fatty acid is inserted into the bilayer,
anchoring the protein to the membrane. If the bilayer is
in the normal fluid state, the fatty acid and its attached
protein can move around in the plane of the bilayer, but
can’t easily leave the bilayer.
Some other lipid-anchored proteins may be anchored
by farnesyl or geranylgeranyl chains covalently
bonded to the C-terminal carboxylate. Farnesyl is a C15
hydrocarbon with several methyl branches,
geranylgeranyl is the C20 branched hydrocarbon chain
The acyl or farnesyl anchored proteins are usually found
on the inside surface of the plasma membrane of the cell
(Lehninger p.400).
GPI anchored proteins are found on the outside of the cell
Certain proteins may be anchored on the exterior surface of the cell, and are linked to the membrane
phospholipid phosphatidyl inositol through a short oligosaccharide chain. This is the
glycosyl-phosphatidyl inositol or GPI anchor. The enzyme PI-PLC (PI specific phospholipase C)
can cut the bond between diacylglycerol and the phosphate, and this can release the protein from its
membrane anchor.
Diacylglycerol-phosphate-inositol-GlcNAc-(Mannose)3 -phosphate-ethanolamine-protein
(in bilayer) ↑
|
C-terminal
PI-PLC can cut here
Mannose (branch)
GlcNAc is the abbreviation for N-acetylglucosamine , in which O-2 of glucose is replaced by NH2 ,
and then an acetyl group attached by an amide bond.
Glycophorin was one of the first integral membrane
proteins studied
Glycophorin is an integral membrane protein from the red blood cell
(Lehninger p.398). Its polypeptide was found to span the bilayer, with
the N-terminal 73 amino acid exposed on the exterior, 21 amino acids
are deeply embedded in the bilayer, and the last 37 amino acids up to the
C-terminus are exposed on the cytoplasmic face of the bilayer. The
number of buried amino acids corresponds to 32 Å in an α-helical
arrangement, and this appears to match the thickness of the hydrocarbon
band of the bilayer.
The N-terminal half of glycophorin has 15 Ser or Thr side chains which are modified by addition of
oligosaccharide chains (O-linked oligosaccharide , i.e. bonded to the O atom of the side chain, shown
as red squares) and one Asn with a different type of N-linked oligosaccharide, bonded to the N atom
of the Asn side chains (blue square in the figure).
It is quite common for proteins that are either destined for secretion or which are exposed on the
outside of the plasma membrane to be extensively modified with oligosaccharide chains. The
oligosaccharide may play a stabilizing role for external proteins, may protect against proteolytic or other
attack, and may provide identity markers to cells which carry them
Integral membrane proteins have different topologies
Topology describes the spatial distribution of different integral or transmembrane proteins.
Membrane proteins are placed in 6 different groups:
Type I is like glycophorin, a single transmembrane helix, N-terminal outside. Type II is also a single
transmembrane helix, but with N-terminal inside. Type III consists of a bundle of helices in a single
polypeptide, which may assemble together to form a transmembrane channel, e.g. for substrate
transport. Type IV may also assemble as a transmembrane channel, but is formed by bringing together
a number of independent helical segments rather than connected as a single polypeptide. Topology
types V and VI (Lehninger p. 401, Fig. 12-14) are actually lipid anchored proteins.
Hydropathy plot
The hydropathy plot is a graph intended to identify transmembrane segments of
proteins by examining the amino acid sequence. This is also known as a
Kyte-Doolittle plot, after the originators of the technique. Protein sequences
may be identified encoded in DNA, and a considerable amount of DNA
sequence information for different organisms is now available.
Each amino acid is given a numerical value called its hydropathy (Lehninger
p.118, Table 5-1, second column from the right), which reflects the amino
acid’s preference for a nonpolar environment.
The sequence is scanned and at each amino acid, the average Hav is calculated
for that amino acid plus 10 neighbours preceeding and 10 neighbours
following. The length of the group that is averaged is called the “window”. A
window of 19 to 23 is considered appropriate for the expected length of the
transmembrane helix.
If the majority of amino acids in the window are nonpolar, as expected for amino acids in contact with
the bilayer, then the average will rise to a positive number. In a random sequence, the average will be
close to zero or slightly negative. Sections of the graph exceed ing a critical threshold value are
predicted to be transmembrane helices (Lehninger p. 403, Fig. 12-17).