Download (Extrinsic) Proteins

Chapters 14 and 16
Membrane Proteins
Channels
Fibrous Proteins
Amyloid Fibrils
Membrane Structure
• Biological membranes are bilipid layers. In a real cell the
membrane phospholipids create a spherical three
dimensional lipid bilayer shell around the cell. However,
they are often represented two-dimensionally as:
• The circle, or head, is the negatively charged phosphate
group and the two tails are the two highly hydrophobic
hydrocarbon chains of the phospholipid. The tails of the
phospholipids orient towards each other creating a
hydrophobic environment within the membrane. This
leaves the charged phosphate groups facing out into the
hydrophilic environment. The membrane is approximately
5 nm thick.
Membrane Properties
• This bilipid layer is semipermeable, meaning that
some molecules are allowed to pass freely
(diffuse) through the membrane.
• The lipid bilayer is virtually impermeable to large
molecules, relatively impermeable to molecules as
small as charged ions.
• It is quite permeable to lipid soluble low
molecular weight molecules. It is substantially
permeable to water molecules. Molecules that can
diffuse through the membrane due so at differing
rates depending upon their ability to enter the
hydrophobic interior of the membrane bilayer.
Membrane Functions
•
•
•
•
•
•
Forms selectively permeable barriers
Transport phenomena
Cell communication and signaling
Cell-cell adhesion and cellular attachment
Cell identity and antigenicity
Conductivity
Membrane-bound Proteins
•
From left to right are (a) a protein whose polypeptide chain
traverses the membrane once as an a helix, (b) a protein that forms
several transmembrane  helices connected by hydrophilic loop
regions, (c) a protein with several b strands that form a channel
through the membrane, and (d) a protein that is anchored to the
membrane by one  helix parallel to the plane of the membrane.
Integral (Intrinsic) Proteins
• penetrate the bilayer or span the membrane entirely
• can only be removed from membranes by disrupting the
phospholipid bilayer
Transmembrane proteins
• Transmembrane proteins
can be divided into two
classes, either (1) singlepass and (2) multiple-pass.
• Trans-membrane proteins
have membrane spanning
portions containing 
helically arranged
sequences of 20-25
hydrophobic amino acids.
Short strings of
hydrophilic amino acids
separate the hydrophobic
sequences from each other.
•These hydrophilic stretches tend to be found exposed to the more aqueous
•environments associated with the cytoplasm or the extracellular space.
Covalently Tethered Integral
Membrane Proteins
• Tethered integral
membrane proteins
may be largely
exposed to either the
cytoplasm or aqueous
extracellular space,
but are covalently
linked to membrane
phospholipids or
glycolipids
Glycoproteins
• Many integral proteins are
glycoproteins covalently
linked to sugars via
asparagine, serine, or
threonine.
• The sugars of
glycoproteins are
exclusively found on the
extracellular side or
topological equivalent of
the membrane.
• Glycosylation begins in
the lumen of the ER
• Carbohydrates are
modified in Golgi
complex.
Properties of Peripheral (Extrinsic) Proteins
• do not penetrate the phospholipid bilayer
• are not covalently linked to other membrane components
• form ionic links to membrane structures
a. can be dissociated from membranes
b. dissociation does not disrupt membrane integrity
• located on both extracellular and intracellular sides of the
membrane and often link membrane to non-membrane structures
synthesis of peripheral proteins:
a. cytoplasmic (inner) side:
made in cytoplasm
b. extracellular (outer) side:
made in ER and undergo
exocytosis
Solubilization and crystallization
of membrane proteins
Bacteriorhodopsin
• An electron density map to 7 Å resolution (a) was obtained and
interpreted in terms of seven transmembrane helices (b). In 1990,
the resolution was extended to 3 Å, which confirmed the presence of
the seven  helices connected by loop regions and where the retinal
molecule was bound to bacteriorhodopsin.
Photoisomerization of Rentinal
• The light-absorbing pigment retinal
undergoes a conformational change
called isomerization when it absorbs
light. One part of the molecule rotates
180° around a double bond between
two carbon atoms (green). The
geometry of the molecule is changed
by this rotation from a trans form to a
cis form.
• Protons are pumped from cytosol to
the extracellular space, creating a
proton gradient.
• The generated proton gradient is used
to produce ATP and to transport ions
and molecules across the membrane.
The Bacteriorhodopsin Proton Channel
and Bound Retinal.
• A to E are the seven
transmembrane helices. Retinal is
covalently bound to a lysine
residue. The relative positions of
two Asp residues, which are
important for proton transfer, are
also shown.
• Retinal is bound in a pocket in the
middle of bacteriorhodopsin.
• It is covalently linked to Lys-216
with a positive charge
• Asp-96 and Asp-85 are located on
both sides of the retinal.
The Photocycle of Bacteriorhodopsin
• The protein adopts two main conformational states, tense (T) and
relaxed (R). The T state binds trans-retinal tightly and the R state
binds cis-retinal. The structure of bacteriorhodopsin in the T state,
shown in (a), with trans-retinal bound to Lys 216 via a Schiff base.
A proton is transferred from the Schiff base to Asp 85 following
isomerization of retinal and a conformational change of the protein,
as shown in (b).
The Photocycle of Bacteriorhodopsin
• The structure of bacteriorhodopsin in the R state with cis-retinal
bound (c). A proton is transferred from Asp-96 to the Schiff base
and from Asp-85 to the extracellular space. A proton is
transferred from the cytoplasm to Asp-96, as shown in (d).
Porin Channels
extracellular
funnel
Hydrophilic
Hydrophobic
periplasm
• The outer membrane of bacteria
contain porin channels with beta
barrels.
• Ribbon diagram of one subunit of
porin from Rhodobacter
capsulatus viewed from within
the plane of the membrane.
Sixteen b strands form an
antiparallel b barrel that traverses
the membrane. The long loop
between b strands 5 and 6 (red)
(eyelet) constricts the channel of
the barrel. Two calcium atoms are
shown as orange circles.
Each Porin Molecule has Three Channels
Hydrophobic
• Large extracellular molecules
are prevented from entering the
channel due to the funnel size of
the trimer.
• Further screening occurs at the
entrance of the channel in the
individual barrels.
• A molecule small enough to
enter the central channel will
then encounter the eyelet where
the charged sidechains determine
the size limitations and the ion
selectivity of the pore.
+
K
Channel
• The membrane potentials of resting cells are determined
largely by K+, which is actively pumped into cells by an
ATP-driven Na, K+ pump.
• Na+ and K+ can also move freely in and out through K+
leak channels.
• The K+ leak channels in the plasma membrane are highly
selective for K+ ions by a factor of 104 over Na+, and have
a throughput of 108 ions per second.
• Why do K+ channels have such high specificity with high
levels of ion conductance?
The Potassium Channel
• Schematic diagram for the
structure of a potassium channel
viewed perpendicular to the plane
of the membrane. The molecule is
tetrameric with a hole in the
middle that forms the ion pore
(purple). Each subunit forms two
transmembrane helices, the inner
and the outer helix. The pore helix
and loop regions build up the ion
pore in combination with the inner
helix.
Potassium Channel Selectivity Filter
• Diagram showing two
subunits of the K+
channel, illustrating the
way the selectivity
filter is formed. Mainchain atoms line the
walls of this narrow
passage with carbonyl
oxygen atoms pointing
into the pore, forming
binding sites for K+
ions.
The Ion Pore of the Potassium Channel
• The cytosolic side of the pore begins as a water-filled channel that opens
up into a water-filled cavity near the middle of the membrane. A narrow
passage, the selectivity filter, links this cavity to the external solution.
Three potassium ions (purple spheres) bind in the pore. The pore helices
(red) are oriented such that their carboxyl end (with a negative dipole
moment) is oriented towards the center of the cavity to provide a
compensating dipole charge to the K+ ions.
Hydropathic Index
Channel selectivity is determined by the conserved
residues at the P-regions.
Glucose Transporters
The glucose binding site alternately
faces the inside and outside of the
cell when occupied by a sugar.
• The thermodynamic
downward movement of
glucose across the plasma
membrane of animal cells
is mediated by several
glucose transporters
(GLUT1-5) composed of
about 500 amino acids.
• The common structural
themes of GLUT is the
presence of 12
transmembrane segments.
The Photosynthetic Reaction Center
• The three-dimensional structure of a
photosynthetic reaction center of a
purple bacterium was the first highresolution structure to be obtained for a
membrane-bound protein. The
molecule contains four subunits: L, M,
H, and a cytochrome. Subunits L and M
bind the photosynthetic pigments, and
the cytochrome binds four heme
groups. The L (yellow) and the M (red)
subunits each have five transmembrane -helices A-E. The H
subunit (green) has one such transmembrane helix, AH, and the
cytochrome (blue) has none.
Approximate membrane boundaries are
shown. The photosynthetic pigments
and the heme groups appear in black.
Photosynthetic Pigments in a
Reaction Center
• Schematic arrangement of the
photosynthetic pigments in
the reaction center of
Rhodopseudomonas viridis.
• The twofold symmetry axis
that relates the L and the M
subunits is aligned vertically
in the plane of the paper.
Electron transfer proceeds
preferentially along the
branch to the right.
Fibrous Proteins
• Fibrous proteins are built up from long fibers.
• Fibrous proteins often form protofilaments or
protofibrils that assemble to structurally specific,
high order filaments and fibrils.
• Dependent on the secondary structure of the
individual molecules, fibrous proteins an be divided
into three classes
• The triple helix (collagen)
• Coil-coil  helices (keretin & myosin)
• b sheet in amyloid fibers and silks
Facilitated Diffusion of Glucose into
Erythrocytes
• All the steps in the transport of glucose into cells are freely
reversible, the direction of movement of glucose being dictated by
the relative concentrations of glucose on either side of membrane.
In order to maintain the concentration gradient across the
membrane, the glucose is rapidly phosphorylated inside the cell to
glucose 6-phosphate by hexokinase.
Collagen
• Collagens are proteins that assemble into fibrous supermolecular aggregates in the extracellular space, which
comprise three polypeptide chains with a large number of
repeat sequences Gly-X-Y where X is often proline and Y
is often hydroxyproline.
• Hydroxyproline is formed by the post-translational
modification of proline by prolyl hydroxylase.
• Each collagen polypeptide chain contains about 1000
amino acid residues and the entire triple chain molecule is
about 3000 Å long.
Types of Collagen
The Collagen Helix
• Each polypeptide chain in
the collagen molecule
folds into an extended
polyproline type II helix
with a rise per turn along
the helix of 9.6 Å
comprising 3.3 residues.
In the collagen molecule,
three such chains are
supercoiled about a
common axis to form a
3000-Å-long rod-like
molecule. The amino acid
sequence contains repeats
of -Gly-X-Y- where X is
often proline and Y is
often hydroxyproline.
Formation of Hydroxyproline & Hydrolysine
Biosynthesis of
Collagen
Gly to Ala Mutation in
Polyproline Type II Helix.
• Models of a collagen-like peptide
with a mutation Gly to Ala in the
middle of the peptide (orange).
• Each polypeptide chain is folded
into a polyproline type II helix and
three chains form a superhelix
similar to part of the collagen
molecule. The alanine side chain is
accommodated inside the superhelix
causing a slight change in the twist
of the individual chains
Hydrogen Bonding in the
Collagen Triple Helix
• In the regular
collagen triple helix,
the three chains are
held close together
by direct interchain
hydrogen bonds
between proline C=O
groups and glycine
NH groups.
Hydrogen Bonds in the Gly-Ala Mutation
• In the region around the
alanine residues, the three
polypeptide chains are forced
apart by the alanine side
chains.
• Four water molecules are
inserted in the interior of the
triple helix to mediate
hydrogen bonds between the
polypeptide chains, which
are displaced due to the
alanine side chains in this
region.
Intrachain Water Bridges in a
Collagen-like Peptide
Space-filling models of intrachain water bridges observed in the crystal
structure of a collagen-like peptide. The oxygen atoms of the water
molecules are yellow and the hydrogen atoms are white. A bridge of three
water molecules hydrogen bonded to two C=O groups and linked to other
water molecules are shown in (a). Long bridges of water molecules
establish a network that surrounds the triple helix shown in (b).
Domain Organization of Intermediate
Filament Monomers
• The domain organization of intermediate filament protein
monomers. Most intermediate filament proteins share a
similar rod domain that is usually about 310 amino acids
long and forms an extended  helix. The amino-terminal and
carboxy-terminal domains are non--helical and vary greatly
in size and sequence in different intermediate filaments.
A Model of Intermediate Filament Construction
• The monomer shown in
(a) pairs with an identical
monomer to form a
coiled-coil dimer (b). The
dimers then line up to
form an antiparallel
tetramer (c). Within each
tetramer the dimers are
staggered with respect to
one another, allowing it to
associate with another
tetramer (d). In the final
10-nm rope-like
intermediate filament,
tetramers are packed
together in a helical array
(e).
Myosin Molecule
• Schematic diagram of the myosin molecule, comprising
two heavy chains (green) that form a coiled-coil tail with
two globular heads and four light chains (gray) of two
slightly differing sizes, each one bound to each heavychain globular head.
Silk Fibers of Spiders
• Individual spiders generate up to seven distinct silk fibers by
drawing liquid crystalline proteins from a set of separate glandspinneret complexes.
• The proteins, silk fibroins, are produced inside the glands from
a family of homologous genes and stored as a highly
concentrated (up to 50%) solution of mainly -helical proteins.
• This solution is passed through the spinning machinery of the
spider and mixed with other components, producing a variety of
different fibers in which the proteins have adopted a b-structure.
• Sequence pattern of silk fibers:
– Variable domains at both N & C-termini flank a large region
of repetitive short sequences of alternating poly-Ala (8 to
10) and Gly-Gly-X repeats (where X is usually S,Y,or Q) up
to 800 residues.
Silk Fibers of Spiders
• Spider fibers are composite
materials formed by large
silk fibroin polypeptide
chains with repetitive
sequences that form b
sheets. Some regions of the
chains participate in
forming 100 nm crystals,
while other regions are part
of a less-ordered meshwork in which the crystals
are embedded. The diagram
shows a model of the
current concepts of how
these fibers are built up,
which probably will be
modified and extended as
new knowledge is gained.
Alzheimer's Disease
• Alzheimer's disease is characterized by the deposition of
insoluble amyloid fibrils as amyloid plaques in the neurophil
as well as the accumulation of neurofibrillary tangles in cell
bodies of neurons.
• The amyloid fibrils are composed of the amyloid b peptide
(Ab), a 39-43 amino acid residue peptide produced by
cleavage from a larger amyloid precursor protein, APP. The
Ab peptide is known to be present in unaffected individuals
and is thought to have a normal physiological role. However,
in Alzheimer's disease patients, the A peptide forms ordered
aggregates which are deposited extracellularly as amyloid
plaques or senile plaques in the neurophil, and as vascular
deposits.
Alzheimer's Disease
• Alzheimer's disease is not the only disease in
which amyloid fibrils are involved in the
pathology. There is now a list of some 16 proteins
which can form amyloid fibrils in various
diseases. These proteins vary considerably in their
primary structure, function, size and tertiary
structure, and yet they appear to form amyloid
fibrils, which show very few structural
differences.
– Immunoglobulin light chain
– Ab-proteins precursor
Electron Micrograph of Amyloid Fibrils
• Electron micrograph of
amyloid fibrils formed in
water from Aβ1-42,
stained with 0.1 %
phosphotungstic acid
showing long straight
fibrils of 70 - 80 Å
diameter as well as fibrillar
aggregate background. Bar
= 1000 Å.
Structure of Amyloid Fibrils
• Electron microscopy has shown that
amyloid fibrils are straight, unbranching fibers of 70-120 Å in
diameter and of indeterminate length.
All amyloid fibrils stain with the dye
Congo red and give a green
birefringence when examined under
cross-polarized light, and finally they
reveal a cross-β fiber diffraction
pattern which suggests a β-sheet type
structure for the fibrils.
Structure of Amyloid Fibrils
• A strong 4.8 Å reflection on the meridian corresponds to the
hydrogen bonding distance between β-strands (shown on the
right), and a more diffuse 10 - 11 Å reflection on the equator
shows the inter-sheet distance of about 10.7 Å. A spacing of
9.6 Å would correspond to the repeat distance for an antiparallel arrangement of β-strands.
Structural Studies of Amyloid Fibrils
• Structural studies of amyloid fibrils from Alzheimer's disease brain
have proved extremely difficult due to the insolubility of the
plaques. Examination of the structure of amyloid fibrils has
concentrated on fibrils formed in vitro from synthetic peptides
homologous to the Aβ peptide.
• However, synthetic amyloid fibrils are problematic to study using
conventional structural techniques. Methods such as single crystal
X-ray crystallography and solution nuclear magnetic resonance
(NMR) cannot be used on fibrils since they are insoluble.
• X-ray fiber diffraction, electron microscopy (EM), solid state NMR,
fourier transform infrared spectroscopy (FTIR) and circular
dichroism (CD) have been used to examine amyloid structure.
The Structure of Soluble Aβ
• Structure prediction studies have indicated that the last 10
residues at the C-terminal and residues 17 - 21 of Aβ show
the greatest hydrophobicity, while the C-terminus (from
residue 28) showed a high probability for β-sheet structure.
Residues 9 - 21showed a lower probability for β-sheet.
Two β-turns are predicted between residues 6 and 8, and
residues 23 and 27.
Structural Studies of Soluble Aβ
• The structure of the Aβ peptide has been extensively
studied in solution, although it is necessary to use
organic solvents such as DMSO and TFE, and
detergents such as SDS to keep Aβ soluble.
• NMR, CD and FTIR studies have shown that Aβ
generally forms -helical conformation in organic
solvents, whereas in aqueous buffers or in water it is
predominantly β-sheet, although this can be affected
by pH, concentration and incubation time.
Conformational Switching
• A b-sheet content is linked to insolubility and related to
neurotoxicity. The fibrillar state is associated with protease
resistance. The A b peptide undergoes a conformation switch from helical to b-sheet structure during amyloidogenesis.
• The N-terminus is thought to be important for initiating
conformational switching. Introduction of a single amino acid
substitution (V18A) in Ab1-40, which leads to an increased
propensity for -helical conformation compared to wild type, was
found to be related to a decreased ability to form amyloid fibrils.
Substitution of residues Lys16 in Ab1-28 or Phe19 or 20 for Ala in
10 - 23 results in peptides unable to form amyloid-like fibrils
• A short peptide 14 - 23 is capable of forming amyloid fibrils in vitro
and the region 11 - 24 is thought to play an important role in the
conversion.
• Residues 25 - 35 have been implicated in neurotoxicity, and residues
34 - 42 are very hydrophobic and are thought to be situated in the
transmembrane region in APP. Peptides of this region are extremely
insoluble and have been implicated in nucleating amyloid fibril
formation.
Structure of Ab Fibril Intermediates
• Beta-sheet conformation is tightly linked to fibrinogenesis
• It is thought that fibril formation proceeds via a nucleation
dependent mechanism, which is highly concentration
dependent and proceeds via a partially folded intermediate.
• Purification of Ab1-42 peptide from the brain of an
Alzheimer's disease patient yielded stable dimeric and
trimeric forms, and monomers that were capable of forming
fibrils in vitro
• Studies of the short Ab14-23 peptide suggest that fibril
polymerization proceeds via the formation of dimers, then
tetramers and finally oligomers, in which the charged
residues form ion pairs and the hydrophobic residues form a
hydrophobic core.
Amyloid Fibrils
• Shown to the left is
the hierarchy of
structure from the Ab
peptide folded into a
b-pleated sheet
structure through
protofilaments to
amyloid fibrils with
b-strands running
perpendicular to the
fiber axis and held
together by hydrogen
bonding.