What are the components of a membrane

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Membrane Proteins

• What are the components of a membrane
• What are the properties of a membrane
• Which are the topologies of membrane proteins
• How to predict the topology
• How to predict the secondary structure

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Cell Membrane
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All molecules move continuously by simple
diffusion
Heat energy causes molecules to move randomly If
the concentration of molecules is different in 2
regions, diffusion will cause molecules to move
from region of high concentration to one of low
concentration
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Hydrophobic Substances Have a High Permeability
Through Bilayer Membranes Hydrophobic
chemicals cross membranes faster than those that
like water Simple diffusion through a
membrane permeability Many biological
chemicals are deliberately made hydrophobic to
increase their rate of penetration into
cells. Examples many drugs, pesticides
such as DDT Energy from ATP is not required
for this type of penetration Hydrophobicity
is measured by oil/water partition The
higher the partition coefficient the higher the
permeability
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It is useful to think of a dilute solution as
having a high water concentration and a
concentrated solution as having a lower water
concentration. Then the water flow goes from
high water to low water concentration.
o In the picture the purple dots represent the
solute (the higher the solute concentration
the lower the water concentration Osmosis
is passive doesn't require ATP energy
Except for the pumping of the blood, all water
movements in the body are by osmosis
Osmotic flow through most biological membranes is
not by simple diffusion- it is by bulk
flow and is similar to the flow caused by a
pressure gradient The kidney is an osmotic
machine it adjusts body water volume by osmosis
Medical problems involving osmosis
pulmonary edema, childhood diarrhea, cholera,
inflammation of tissues
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In the drawing an extracellular molecule binds
to the transport protein the transport protein
then rotates and releases the molecule inside
the cell Examples o Glucose
transporters- 5 different GLUT proteins and 2
types that cotransport Na and glucose
(these are used for secondary active
transport) o Water channels- 8
different types of aquaporins Properties of
facilitated diffusion Facilitated diffusion
cannot cause net transport of molecules from a
low to a high concentration this would
require input of energy ATP energy not
required Transport rate reaches a maximum
when all of the protein transporters are being
used (saturation) Very specific allows
cell to select substances taken up
Sensitive to inhibitors that react with protein
side chains
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Transmembrane proteins
A transmembrane protein is a protein that spans
the entire biological membrane. Transmembrane
proteins aggregate and precipitate in water.
They require detergents or nonpolar solvents for
extraction, although some of them (beta-barrels)
can be also extracted using denaturing agents.
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Why they are important?
Some estimated that around 30 of our genes are
transmembrane. Functionally critical Very easy to
predict sec. str. due to distinct composition of
membrane proteins (gt95) Not many structures
known, a good application for bioinformatics
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There are two basic types of transmembrane
proteins 1. Alpha-helical. These proteins
are present in the inner membranes of bacterial
cells or the plasma membrane of eukaryotes, and
sometimes in the outer membranes. This is the
major category of transmembrane proteins. 2.
Beta-barrels. These proteins are so far found
only in outer membranes of Gram-negative
bacteria, cell wall of Gram-positive bacteria,
and outer membranes of mitochondria and
chloroplasts. All beta-barrel transmembrane
proteins have simplest up-and-down topology,
which may reflect their common evolutionary
origin and similar folding mechanism. Another
classification refers to the position of the N-
and C-terminal domains. Types I and II are single
pass molecules, with the distinction that the
type I transmembrane proteins have their
N-terminal domains targeted to the ER lumen
during synthesis (and the extracellular space, if
mature forms are located on plasmalema), while
type II have their N-terminal domains targeted to
the cytoplasm. Types III and IV are multi-pass
proteins. Types I and III are subdivided into Ia
and Ib, and IIIa and IIIb respectively, depending
on the presence or absence, respectively, of SRP,
the translocation signal sequence. The
implications for the division in type I and II
are especially manifest at the time of
translocation and ER-bound translation, when the
protein has to be passed through the ER membrane
in a direction dependent on the type.
from WIKIPEDIA
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A attachment by ionic interactions between the
protein and the cytosolic face of the lipid
bilayer B attachment via lipid added
posttranslationally and inserted into the
cytosolic leaflet of the bilayer C,D
transmembrane proteins have a part of chain
embedded in the lipid bilayer Cbitopic the
protein chain crosses the membrane once only. D
polytopic membrane proteins, the protein chain
threads back and forth across the membrane
multiple times.
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Two classes of integral membrane proteins
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Beta barrels porins
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Folding pathway of porins

It was shown that integral membrane proteins of
the ß-barrel type, for instance porin of the E.
coli outer membrane, can be fully denatured in 8
M urea. These proteins will spontaneously insert
and refold when the urea is strongly diluted by
mixing with a large volume of urea-free buffer
containing lipid vesicles in the
liquid-crystalline phase (Surrey Jähnig, 1992).
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Membrane Proteins- positive aspects Involved
in lots of processes transport and transduction
processes that mediate the flow of matter
energy and information across the membrane
bilayer poorly characterized relative to
water-soluble proteins experimental challenges
of mimicking the membrane and water-bilayer interf
aces constitute an estimated 20-30 of all
proteins, yet only100 distinct structures are
available (116 as of 10/25/06 133 as of
10/17/07) 50 of drug targets
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http//blanco.biomol.uci.edu/Membrane_Proteins_xta
l.html
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Water permeation through aquaporin de Groot, B.
L. Grubmuller, H. Water permeation across
biological membranes mechanism and dynamics of
aquaporin-1 and Glpf. Science 294 2353-2357,
2001 http//www.mpibpc.mpg.de/abteilungen/073/ga
llery.html
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Prof Grubmuller
http//www.mpibpc.mpg.de/abteilungen/073/gallery.h
tml
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Membrane protein assembly
Constitutive membrane proteins, i.e. those that
are encoded in a normal cells genome and are
responsible for vital physiological activities,
are assembled by means of a complex process
involving synthesis of membrane proteins by
ribosomes attached transiently to a complex of
proteins referred to as a translocon located in
the membrane of the ER. This translocon provides
a transmembrane tunnel into which the newly
synthesized protein can be injected. After
synthesis is complete, the ribosome disengages
from the translocon (which enters a closed state)
and the protein is released into the membrane
bilayer where it assumes (in an unknown way) its
final folded three-dimensional structure.
http//blanco.biomol.uci.edu/Bilayer_Struc.html
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History
T. F. Smith and M. S. Waterman. The
identification of common molecular subsequences,
J. of Molecular Biology, 147195--197,
1981. Kyte, J., Doolittle, R.F., A simple method
for displaying the hydropathic character of a
protein, J. Mol. Biol., 157(1)105--132,
1982. http//fasta.bioch.virginia.edu/fasta/grease
.htm
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Hydropathy plots and the hydrophobic moment -
identification of transmembrane segments of
proteins. Hydropathy is a numeric
representation of the tendency of each amino acid
residue to enter a non-polar environment. A
sequence is scanned in linearly and a moving
average of hydropathy values is computed. J.
Kyte R.F. Doolittle, J. Mol. Biol. 157, 105-132
(1982). Experimental Thermodynamic scales e.g.
measurement of free energy of transfer of amino
acid from oil phase to water
Keq A.A. (np) A.A. (aq) ?G -RT ln Keq
(hydrophobic will give positive values) Nozaki
Tanford, J.Biol. Chem. 246, 2211-2217
(1971). This scale gives high value for Trp and
Tyr due to large surface area, and a low value
for Ala. Statistical scales e.g. Janin, Nature
277, 491 (1979) Sweet and Eisenberg, J. Mol.
Biol 171 479 (1983) Engleman and Steitz, Ann.
Rev. Biophys. Biophys. Chem. 15 321 (1986) are
based on mean of surface area of each amino
acid buried in protein interior (or bilayer
interior) from compilations of existing
structural data. There is significant conflict
between thermodynamic and statistical scales!
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A hydrophilicity plot is a quantitative analysis
of the degree of hydrophobicity or
hydrophilicity of amino acids of a protein. It
is used to characterize or identify possible
structure or domains of a protein. http//fasta.bi
och.virginia.edu/fasta_www2/fasta_www.cgi?rmmisc1
The plot has amino acid sequence of a protein
on its x-axis, and degree of hydrophobicity and
hydrophilicity on its y-axis. The
Kyte-Doolittle scale indicates hydrophobic amino
acids, while the Hopp-Woods scale measures
hydrophilic residues. Analyzing the shape of the
plot gives information about partial structure of
the protein. For instance, if a stretch of about
20 amino acids shows positive for hydrophobicity,
then this indicates that these amino acids may
be part of alpha-helix spanning across a lipid
bilayer, which is composed of hydrophobic fatty
acids. Conversely, amino acids with high
hydrophilicity indicate that these residues are
in contact with solvent, or water, and that they
are therefore likely to reside on the outer
surface of the protein.
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Transmembrane ? Hydrophobic alpha helix
Transmembrane regions are generally predicted
looking for hydrophobic regions of a specific
length in the sequence.
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Transmembrane helices are visible in structures
of membrane proteins determined by X-ray
diffraction. They may be also predicted on the
basis of hydrophobicity. Because the interior of
the bilayer and the interiors of most proteins
of known structure are hydrophobic, it is
presumed to be a requirement of the amino acids
that span a membrane that they be hydrophobic as
well. However, membrane pumps and ion channels
also contain numerous charged and polar residues
within the generally non-polar transmembrane
segments. Using hydrophobicity analysis to
predict transmembrane helices enables a
prediction in turn of the "transmembrane
topology" of a protein i.e. prediction of what
parts of it protrude into the cell, what parts
protrude out, and how many times the protein
chain crosses the membrane. Such prediction
methods are commonly applied with a limited
success.
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Simple sliding window
The hydrophobic patch is so prominent that simple
scanning window for such region is used for TM
prediction (Kyte Doolittles)
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Aggregate scales Kyte Doolittle and Eisenberg
et al. J. Mol. Biol. 179, 125-142 (1984)
average out many existing published scales.
Criticism this eliminates distinctive features
of a particular scale that may be applicable and
optimized for a specific circumstance. Significa
nce of "window" length in hydropathy plots The
hydropathy plot is based on a moving average H(n)
spanning a "window" of 2n 1 amino acids. The
original Kyte Doolittle paper used a window of
7 (n 3), with the intention that the
researcher use judgment.
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Multi-helix membrane proteins
With proteins containing more than one
transmembrane segment one has to predict Which
segments will form the helices The relative
orientation of the helices in the membrane Side
chain orientations
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A helical wheel is a type of plot or visual
representation used to illustrate the properties
of alpha helices in proteins. The sequence of
amino acids that make up a helical region of the
protein's secondary structure are plotted in a
rotating manner where the angle of rotation
between consecutive amino acids is 100, so that
the final representation looks down the helical
axis. The plot reveals whether hydrophobic
amino acids are concentrated on one side of the
helix, usually with polar or hydrophilic amino
acids on the other. This arrangement is common
in alpha helices within globular proteins, where
one face of the helix is oriented toward the
hydrophobic core and one face is oriented toward
the solvent-exposed surface. Specific patterns
characteristic of protein folds and protein
docking motifs are also revealed, as in the
identification of leucine zipper dimerization
regions and coiled coils.
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The helices contain both hydrophobic and charged
residues forming a structural element that has a
different character on each side an amphipathic
helix
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A measure of the amphipatic nature of a helix is
the hydrophobic moment the hydrophobicity of a
peptide measured for different angles of rotation
per residue. It is calculated for all angles of
rotation from 0 to 180 degrees. The hydrophobic
moment identifies when the residues on one side
of the structure are more hydrophobic than on
the other. It is sensitive enough to distinguish
between transmembrane helices and amphipathic
helices in globular protein domains. Surface
helices have, in general, high hydrophobic moment
while membrane helices have low hydrophobic
moment and high hydrophobicity, and hydrophilic
helices have low hydrophobic moment and low
hydrophobicity.
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Three-dimensional structure of bovine rhodopsin
Greenproline residues cause distorsions in the
helices
Schematic representation of the arrangement of
rhodopsin in the membrane.
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Knowledge-based prediction results for rhodopsin
obtained by SOSUI. http//bp.nuap.nagoya-u.ac.jp/s
osui/about-sosui.html Physicochemical properties
of amino acid sequenceshydrophobicity and
charge. A membrane protein must have at least one
very hydrophobic primary transmembrane helix
other transmembrane helices can exist but their
hydrophobicity can be similar to hydrophobic
segments found in soluble proteins. The primary
transmembrane helices are stabilized by a
combination of amphiphilic side chains at the
helix ends as well as having an hydrophobic
central region. Four parameters used hydropathy
index, amphiphilicity index, aminoacid charges
index, each sequence length.
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HMMTOP prediction for rhodopsin
Positive-inside rule (von Heijne 1992) the
intracellular loops between transmembrane
helices have a higher content of arginines R and
lysines K than do extracellular groups. This
rule reflects the observation that nonmembrane
regions inside have more positively charged than
the regions outside the cell.
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TopPreds Hydrophobicity
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TopPred Topology.

• Found 2 segments
• Candidate membrane-spanning segments
• Helix Begin - End Score Certainity
• 1 28 - 48 1.311 Certain
• 2 52 - 72 1.272 Certain
• Total of 1 structures are to be tested
• HEADER START STOP LEN PROB HP DARGLYS
  DCYTEXT DNCHARGE DNNEGPOS
• TOPOLOGY 1 1.00 3.00
  0.07 1.00 0.00
• TOPOLOGY N-in
  N-out N-in
• CYT_LOOP 1 27 27 3.00
  ( 0.80)
• TRANSMEM 28 48 21 1.00 1.31
• EXT_LOOP 49 51 3 1.00
  ( 0.05)
• TRANSMEM 52 72 21 1.00 1.27
• CYT_LOOP 73 214 142 ( 25.00)
  0.07
• //

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Single and Multiple sequences
Single sequence input prediction 77 success
rate Multiple alignment input 86 (Rost, 1996)
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First Topology Prediction Algorithm1992 Von
Heijine
TopPred http//www.ch.embnet.org/software/TMPRED_
form.html http//www.sbc.su.se/miklos/DAS/ http/
/www.enzim.hu/hmmtop/ http//www.cbs.dtu.dk/servic
es/TMHMM-2.0/ http//phobius.sbc.su.se/
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Transmembrane strands (shaded blue, according to
Xray determination) Aromatic residues (bold)
flanking the beta-strands. Last line prediction
with the program PROFtmb using Eisenberg
hydrophobicity scale
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The sequence of the beta-strands have a
hydrophobic residue at every second position in
the sequence the outside cylinder will be
non-polar and the inside polar
Hs(i) 1/4h(i-2) h(i) h(i2) h(i 4)
If the residue at i-2 or i-4 is aromatic, the
hydrophobicity value is increased to 1.6. This
biases the prediction towards strands with
aromatic residues at the end
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TMAP 1997 (Persson Argos)

• 12 kinds of amino acids ? Head or Tail
• Single or multiple sequences

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TMHMM 1998 (Sonhammer etc)

• TMHMM made by the Hidden Markov Experts
• Reported accuracy 77 with 83 structures.
• Biological pattern is well represented by HMMs.

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Architecture
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http//www.cbs.dtu.dk/services/TMHMM/
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Difficulty in topology prediction
The importance of these regulated interactions
is illustrated by analysis of topology prediction
algorithm failures. Misassigned or misoriented TM
domains occur because the primary sequence and
overall hydrophobicity of a single TM domain are
not the only determinants of membrane
integration. J Cell Sci 2002 May 15115(Pt
10)2003-2009. Integral membrane protein
biosynthesis why topology is hard to
predict.Ott CM, Lingappa VR.
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WWW sites for Protein secondary structure
predictionPredict-protein server - one of the
best www.predictprotein.org/ and
cubic.bioc.columbia.edu/predictprotein/See
also http//genomic.sanger.ac.uk/pss/pssb.html
and http//www.cmpharm.ucsf.edu/nomi/nnpredict.
htmlLocal Garnier/Osguthorpe/Robson site
http//fasta.bioch.virginia.edu/fasta/garnier.htm
Local Chou-Fasman site http//fasta.bioch.virgini
a.edu/fasta/chofas.htmMembrane helix
predictionKyte-Doolittle hydropathy plot
http//fasta.bioch.virginia.edu/fasta/grease.htmT
MPred program http//www.isrec.isb-sib.ch/softwar
e/TMPRED_form.htmlClassification and Secondary
Structure Prediction of Membrane Proteins (SOSUI)
http//www.tuat.ac.jp/mitaku/adv_sosui/
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Praline Jaap Heringa Amsterdam
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In summary

• Predict the secondary structure is very easy
• Predict helix orientation is easy
• Predict the number of helices is relatively easy
• What about the loops? they have a functional
  role.
• Molecular modelling caution!!! Too few
  structures still!

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Learning outcomes
What are membrane proteins and why they are
useful Principal Topologies Hydrophobicity
scales Helical Wheels diagrams Positive inside
out rule Secondary structure prediction