Membrane Proteins

• Proteins that interact with biological membranes.

• Targets of over 50% of all modern medicinal drugs.
• Approximately 20–30% of all genes in most genomes encode
membrane proteins.

Overington JP, Al-Lazikani B, Hopkins AL (December 2006);  Krogh, A.; Larsson, B. R.; Von Heijne, G.; Sonnhammer, E. L. L. (2001);
http://drugdiscoveryopinion.com/2009/02/membrane-proteins-anchored/
• Serve as mediators between cell and the extracellular
region, or interior of an organelle and the cytosol.
• Transport metabolites and ions across the cell membrane.
• Convert energy from light into chemical and electrical
energy, and couple flow of electrons to ATP synthesis.
• Act as signal receptors and transduce signals across
membrane (e.g., neurotransmitters, growth factors,
hormones, light or chemotactic stimuli).
• Perform various enzyme functions.
• Cell recognition and communication.
 Membrane Protein Classifications
• Integral membrane proteins
– These are permanently attached to membrane
• Lipid-linked proteins
– These are covalently bonded to a fatty acid such as
palmitate or myristate and serves to anchor the
protein to the cell membrane.
• Peripheral membrane proteins
– These are temporarily attached to membrane or
to integral membrane proteins
Fluid Mosaic Model of Lipid Bilayer
 Crystallization of Membrane Proteins
• Difficult to crystallize because:
– Insoluble in aq. Buffers (Due to hydrophobic surface regions)
– Tend to denature in organic solvents
• Can be solubilized using aqueous detergents, then purified in
native state.

How this works:

Amphipathic detergent adheres to
hydrophobic surface of protein, so
protein-detergent complex
becomes hydrophilic.
Can be crystallized in this state,
but frequently produces low
resolution crystals

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1988/illpres/crystals.html
 2D Crystals of Membrane Proteins
• 2D crystals of membrane proteins can be studied using electron microscopy.
• Bacteriorhodopsin is a 248 residue, integral membrane protein that binds retinal and
uses light energy to transport protons across the membrane.
• Bacteriorhodopsin was first visualized using this method in 1976 (7 Å resolution)
– Observed to have 7 transmembrane α-helices.
– Helices confirmed in 1990 at 3 Å resolution.
– Structure now available at 2 Å resolution.
• Structural studies of bacteriorhodopsin provided information used to predict
transmembrane helices from primary sequence.

http://www.bio.miami.edu/tom/courses/bil255/bil255goods/12_membrane.html
 Integral Membrane Proteins
• Functions
– Transfer information
– Transport material
– Energy conversion
• Types
– Type I: Single transmembrane span, N-terminus in ectodomain,
C-terminus in cytosol
– Type II: Single span, C-terminus in the ectodomain, N-terminus
in the cytosol
– Type III: Multiple spans
– Type IV: Several different polypeptides assembled to form a
channel
– Type V: Lipid-linked protein
– Type VI: Proteins with both transdomain component and lipid
anchor
Nelson, D. L., & Cox, M. M. (2008). Principles of Biochemistry (5th ed., p. 377).
 Integral membrane proteins - glycoproteins
• Proteins that contain oligosaccharide chains (glycans)
covalently attached to AA side chains as co- or post-
translational (N- or O-) glycosylation.
• Function as receptors.
 Integral membrane proteins - Sialoglycoproteins
• Glycoproteins combined with sialic acid.
• Glycophorins (A –D) are sialoglycoproteins found in the
membranes of red blood cells.

Glycophorins A and B: Bear antigenic

determinants for MN and Ss blood
groups.

Glycophorins C and D: Maintains

erythrocyte shape and regulates
membrane properties. Acts as receptor
for Plasmodium falciparum protein.

1afo
 Porins/Ion Channels
Porins
• 16 Strand, antiparallel up and down β-barrel proteins that form trimer to
traverse the cell membrane.
• Sequence composed of alternating hydrophobic and hydrophilic residues:
hydrophobic residues face the lipid membrane, hydrophilic residues form
aqueous channels for molecules to pass through the membrane.

The porin channel

• Channel blocked by loop (the eyelet)
which limits size of solute that can pass
• Channel is lined with charged amino
acids (positive on 1 side, negative on
the other) – Ion selectivity.
• Calcium ions bind in channel to
balance excess negative charges
• Found in outer membrane of Gram-
negative bacteria, mitochondria and
the chloroplast

http://commons.wikimedia.org/wiki/File:Sucrose_specific_porin_1A0S.png#mediaviewer/File:Sucrose_specific_porin_1A0S.png
 Ion/Potassium Channels
• Allow potassium, sodium,
calcium and chloride ions to
diffuse across lipid bilayer to
balance differences in electrical
charge (membrane potential)
between the 2 environments.
• The membrane potential of
resting cells is regulated
primarily by potassium ions
moving through potassium
channels.

Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001)

 Potassium Channels - Overview
• Most widely distributed type of ion channel
• Found in virtually all living organisms and most types of cells
• Span cell membranes to allow passage of K+ into cells
• Potassium channels conduct potassium ions down their electrochemical gradient,
doing so both rapidly (up to the diffusion rate of K+ ions in bulk water) and selectiv
ely (by a factor of 10, 000 over Na+, despite the sub-angstrom difference in ionic ra
dii).
• Biologically, these channels act to set or reset the resting potential in many cells.
• In excitable cells, such as neurons, the delayed counterflow of potassium ions
shapes the action potential.
• By contributing to the regulation of the action potential duration in cardiac muscle,
malfunction of potassium channels may cause life-threatening arrhythmias.
Potassium channels may also be involved in maintaining vascular tone.
• They also regulate cellular processes such as the secretion of hormones (e.g.,
insulin release from beta-cells in the pancreas) so their malfunction can lead to
diseases (such as diabetes).
 Potassium Channels - Types
• Calcium-activated potassium channels - Open in
response to the presence of calcium ions or other
signalling molecules.
• Inwardly rectifying potassium channels – Pass positively-
charged current more easily into the cell.
• Tandem pore domain potassium channel - "leak
channels" regulated by oxygen tension, pH, mechanical
stretch, and G-proteins. These set the negative
membrane potential of neurons.
• Voltage-gated potassium channels - Open or close in
response to changes in the transmembrane voltage.
 Calcium-activated potassium channels

Based on conductance (big, intermediate, and small),

can be sub-divided into:
• BK channels
• IK channels
• SK channels
 Calcium-activated BK potassium channels
• BK channels (Big Potassium), also called slo1, are characterized by large
conductance of potassium ions (K+) through cell membranes.
• Channels are activated by changes in membrane electrical potential
and/or by increases in intracellular [Ca2+].
• Essential for the regulation of several physiological processes including
smooth muscle tone and neuronal excitability.

A: structure of the α and β1-subunits of the BK channel. The β1-subunit consists of 2

transmembrane domains and the α-subunit of 11 (S0–S10) hydrophobic domains, with
S0–S6 located in the cytoplasmic membrane and the pore region (P) between S5 and S6.
B: Association of four α and four β1-subunits forms the native BK channel.
An overview of the potassium channel family, Miller, 2000; Calcium-Activated Potassium Channels and the Regulation of Vascular Tone, Ledoux
et al., 2006
 Calcium-activated IK potassium channel
• The IK channel (KCa3.1) is expressed mainly in peripheral tissues such as
those of the haematopoietic system, colon, placenta, lung and pancreas.
• Has been implicated in a wide range of cell functions, including
vasodilation of the microvasculature, K+ flux across endothelial cells of
brain capillaries and phagocytic activity of neutrophils.
• In comparison with the large-conductance (BK) channels, KCa3.1 is much
more sensitive to Ca2+ than BK channels.
– This high affinity is due to the presence of 4 calmodulin molecules bound to the
cytoplasmic tails of the four pore-forming α-subunits.
– Before the channel can open, Ca2+ must bind to each of the calmodulins to induce the
co-operative conformational change that opens the gate.

Cross-section of the IK channel. Binding of

calcium ion to CaM produces conformational
change that opens the pore.

http://www.biochemj.org/csb/003/csb003fig3_IK_and_SK_channel_opening.htm
 Calcium-activated SK potassium channels
• Small single channel conductance .
• SK channels allow potassium ions to cross the cell membrane and are activated (opened) by
an increase in the intracellular [Ca 2+] through N-type calcium channels.
• SK channels are thought to be involved in synaptic plasticity and therefore play important
roles in learning and memory.

SK channels are Ca-activated K channels, gated solely by intracellular Ca ions. SK channels are
constitutively associated with calmodulin (CaM) that binds to the CaMBD in the intracellular C-
terminus of the channels. When Ca ions bind to the N-lobe E-F hands of CaM, a gating transition
is initiated, the gate of the channel opens and K flows through the channel pore, exerting a
repolarizing influence on the membrane potential.
Calcium-Activated Potassium Channels and the Regulation of Vascular Tone, Ledoux et al., 2006; http://www.ohsu.edu/xd/research/centers-
institutes/vollum/faculty/adelmanlab.cfm
 Inwardly rectifying potassium channels
• There are seven subfamilies of Kir channels,
denoted as Kir1 - Kir7. Each subfamily has
multiple members that have nearly identical
AA sequences across known mammalian
species.
• Kir channels are formed as homotetrameric
membrane proteins. Each of the 4 identical
protein subunits consists of 2 membrane-
1p7b spanning alpha helices (M1 and M2).

• The voltage-dependent block by polyamines

(i.e., spermine, spermadine) and
magnesium ions from within the cell directs
positive current (K+) inwards.
• This current may be involved in regulation
of neuronal activity, by stabilizing resting
membrane potential of the cell.
• This mechanism remains to be determined.

Structural insights into gating and the formation of a macromolecular GIRK signalling complex, Nature Reviews, 2010;
 Tandem pore domain potassium channel
• Tandem pore domain potassium channels are a family of 15 members form what is known as
"leak channels“.
• Leak channels allow potassium to exit the cell in order to reduce positive charge within the cell
and maintain stable resting membrane potential.
• Channels are regulated by several mechanisms including:
– oxygen tension
– pH
– mechanical stretch
– G-proteins
• For most, the α subunits consist of 4 transmembrane segments, each containing 2 pore loops.
• They structurally correspond to two inward-rectifier α subunits and thus form dimers in the
membrane.

K2P dimer

http://swift.cmbi.ru.nl/gv/students/prachi/website/Potassium_channel_4TM.html
 Voltage-gated potassium (Kv) channels - Overview
• Present in all animal cells.
• Open and close based on voltage changes in the cell's membrane potential.
• During action potentials, these channels open and allow passive flow of K + ions
from the cell to return the depolarized cell to a resting state.
• Kv channels are one of the key components in generation and propagation of
electrical impulses in nervous system.

http://www.ks.uiuc.edu/Research/kvchannel/; Voltage-gated potassium channels as therapeutic targets, Wulff et al., 2009

 Voltage-gated potassium (Kv) channels - Overview
• The tetrameric structure of Kv channels is made of two functionally and structurally
independent domains: an ion conduction pore, and voltage-sensor domains.
• The ion conduction pore is made of four subunits which are arranged symmetrically around
the conduction pathway.
• Voltage-sensor domains are positioned at the periphery of the channel and consist of four
transmembrane segments (S1-S4).
• Structural rearrangement of the voltage-sensor domains in response to changes in the
membrane potential, and in particular S4, which includes positively charged amino acids at
every third position, results in conformational changes in the conduction pore, which could
open or close the ion conduction pathway.
• The nature of these movements and conformational changes in the voltage-sensors have
been subject to controversy and several models for voltage-gating have been proposed.

Ion channels: A paddle in oil, Lee, 2006; http://www.ks.uiuc.edu/Research/kvchannel/

 Calcium channels - Overview
Two types of ion channels with selective permeability to calcium ions
I. Voltage gated calcium channels
I. L-type (High voltage activated)
I. Found in muscle, bone, ventricular myocytes
II. P-type (High voltage activated)
I. Found in Purkinje neurons in cerebellum
III. N-type (Hight voltage activated)
I. Found in brain and peripheral nervous system
IV. R-type (Intermediate voltage activated)
I. Found in cerebellar granule cells, other neurons
V. T-type (Low voltage activated)
I. Found in neural cells, bone
II. Ligand gated calcium channels
I. IP3 receptor
II. Ryanodine receptor
III. Two pore channels
IV. Cation channels of sperm
V. Store operated channels

Ion channels: A paddle in oil, Lee, 2006; http://www.ks.uiuc.edu/Research/kvchannel/

 L-type high-voltage-activated Calcium channels
• L-type calcium channels remain activated for longer periods of time.
• Channel is comprised of four protein subunits (Cav1.1, Cav1.2, Cav1.3, Cav1.4).
• Responsible for excitation-contraction coupling of skeletal, smooth, cardiac muscle
and for aldosterone secretion in endocrine cells of the adrenal cortex.
• In cardiac myocytes, the L-type calcium channel direct Ca2+ current into the cell
and triggers calcium release from the sarcoplasmic reticulum (SR) by activating
Ryanodine receptor 2 (RyR2).

Voltage-gated calcium channels in the human adrenal and primary aldosteronism. J Steroid Biochem Mol Biol., Felizola et al., 2014; Calcium
channels — basic aspects of their structure, function and gene encoding; anesthetic action on the channels — a review. Can J Anaesth.,
Yamakage M, Namiki A, 2002
 L-type high-voltage-activated Calcium channels
• The alpha-1 subunit forms the pore for
the import of extracellular calcium ions
and, though regulated by the other
subunits, is primarily responsible for
the pharmacological properties of the
channel [PMID: 11031246].
• It shares sequence characteristics with
all voltage-dependent cation channels,
and exploits the same 6-helix bundle
structural motif - in both sodium and
calcium channels, this motif is repeated
4 times within the sequence to give a
24-helix bundle. Within each of these
repeats, 5 of the transmembrane (TM)
segments (S1, S2, S3, S5, S6) are
hydrophobic, while the other (S4) is
positively charged and serves as the
voltage-sensor.

Calcium channels — basic aspects of their structure, function and gene encoding; anesthetic action on the channels — a review. Can J Anaesth.,
Yamakage M, Namiki A, 2002; Voltage-Gated Calcium Channel Antagonists and Traumatic Brain Injury, Gurkoff et al., 2013
 IP3 Receptor
• Inositol trisphosphate (IP3) receptor is a membrane glycoprotein complex acting as
a Ca2+ channel activated by inositol trisphosphate (InsP3).
• IP3R is necessary for the control of cellular and physiological processes including:
– cell division
– cell proliferation
– apoptosis
– fertilization
– development
– behavior
– learning
IP3 binding core (IBC) of IP3
– memory receptor with bound IP3 molecule
• IP3R represents a dominant second messenger leading to the release of Ca 2+ into
the cytosol from intracellular store sites (i.e., SR lumen).

Acting on a membrane phospholipid,

phospholipase C cleaves off IP3, which is
a small polar molecule.
IP3 binds to IP3R which opens to release
calcium into the cytosol.

Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Bosanac I, Alattia JR, Mal TK, et al., Nature, 2002;
http://courses.washington.edu/conj/gprotein/ip3.htm
 Ryanodine Receptor 1
• RYR1 functions as an SR calcium release channel, as well as a connection between
the sarcoplasmic reticulum and the transverse tubule.
• Found primarily in skeletal muscle.
• Triggers muscle contraction following depolarization of T-tubules.
• Repeated very high-level exercise increases the open probability of the channel
and leads to Ca2+ leaking into the cytoplasm.
• Can also mediate the release of Ca2+ from intracellular stores in neurons, and may
thereby promote prolonged Ca2+ signaling in the brain.
• Required for normal embryonic development of muscle fibers and skeletal muscle.
• Required for normal heart morphogenesis, skin development and ossification
during embryogenesis.

http://www.uniprot.org/uniprot/P21817
 Lipid-linked proteins: G proteins
• G proteins, (guanine nucleotide-binding proteins), are a family of proteins that act
as molecular switches inside cells.
• Transmit signals from a variety of stimuli outside a cell to the inside.
• Their activity is regulated by factors that control their ability to bind to and
hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP).
• When they bind GTP, they are 'on', and, when they bind GDP, they are 'off'.
• G proteins belong to the larger group of enzymes called GTPases.
(1) ligand activates the G protein-coupled
receptor. (2) Induces a conformational
change in the receptor that allows the
receptor to function as a guanine
nucleotide exchange factor (GEF) that
exchanges GTP for GDP (4) - thus turning
the GPCR "on". The GTP (or GDP) is bound
to the Gα subunit in the traditional view
of heterotrimeric GPCR activation. (5)This
exchange triggers the dissociation of the
Gα subunit (which is bound to GTP) from
the Gβγ dimer and the receptor as a
whole.

Gα subunit stimulates production of

cAMP from ATP.
http://en.wikipedia.org/wiki/G_protein
 Peripheral membrane proteins
• Adhere only temporarily to the biological membrane with which they are
associated (reversible binding).
• Attach to integral membrane proteins, or penetrate the peripheral regions of the
lipid bilayer.
– May be regulatory protein subunits of many ion channels and transmembrane
receptors.
• Tend to collect in the water-soluble component, or fraction, of all the proteins
extracted during a protein purification procedure.
– Proteins with GPI anchors are an exception to this rule and can have
purification properties similar to those of integral membrane proteins.
• Reversible binding of these proteins is associated with regulation of cell signaling
and other cellular events.
• These proteins may interact with membrane in several ways:

1. By amphipathic α-helix parallel to
membrane
2. By a hydrophobic loop
3. By a covalently bound membrane lipid
(lipidation)
4. By electrostatic or ionic interactions
 with membrane lipids (e.g. through a
calcium ion)

http://en.wikipedia.org/wiki/Peripheral_membrane_protein
 Categories of peripheral membrane proteins
• Enzymes
– Phospholipase C: Hydrolyzes PIP2 into IP3 and diacylglycerol
• Membrane-targeting domains (lipid clamps)
– C2 domains: Bind phosphatidylserine or phosphatidylcholine
• Structural domains
– Annexins: Calcium-dependent intracellular membrane/phospholipid binding. Involved in
membrane fusion and ion channel formation.
• Transporters of small hydrophobic molecules
– Glycolipid transfer protein
• Electron Carriers
– Cytochrome C: Transfers electrons between Complex III (CoQ-Cyt C reductase) and
Complex IV (Cyt C oxidase) in the electron transport chain.
• Polypeptide hormones, toxins and antimicrobial peptides
– Venom toxins (e.g., snake venom)

http://en.wikipedia.org/wiki/Peripheral_membrane_protein
 Phospholipase C

• Enzymes that cleave phospholipids just before the phosphate group.

• PLC cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)
into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).
• DAG remains bound to the membrane.
• IP3 is released as a soluble structure into the cytosol, where it binds to IP3
receptors in the smooth endoplasmic reticulum (ER), releasing calcium
into cytosol, causing a cascade of intracellular changes.
• Calcium and DAG also work together to activate protein kinase C, which
goes on to phosphorylate other molecules, leading to altered cellular
activity.
"PLC role in IP3-DAG pathway" by RaihaT - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons -
http://commons.wikimedia.org/wiki/File:PLC_role_in_IP3-DAG_pathway.tif#/media/File:PLC_role_in_IP3-DAG_pathway.tif
 C2 Domains
• Protein structural domains that help target proteins to cell membranes.
• Typical structures consist of a beta-sandwich with 8 strands that may bind
2-3 calcium ions.
• C2 domains frequently bind with enzyme domains.
– Example: The C2 domain in PTEN, brings the phosphatase domain into contact with the
plasma membrane, where it can dephosphorylate its substrate, phosphatidylinositol
(3,4,5)-trisphosphate (PIP3), without removing it from the membrane.

Synaptotagmin binds phospholipids in a calcium

dependent manner. Synaptotagmin's C2 domain
shows a single calcium-binding site made up of
residues from two loops at one end of the
domain (red spheres). PI-PLC delta 1 also has
calcium binding between the analogous loops
(cyan spheres). However, PI-PLC has two
prominent calcium binding sites in this location.
The presence of these sites and the orientation
of this domain with respect to the catalytic
domain suggest that the C2 domain may be
important for interacting with phospholipid
head groups of the membrane.

http://en.wikipedia.org/wiki/C2_domain; http://www.mrc-lmb.cam.ac.uk/rlw/text/c2domain.html
 Annexins
• Over 160 different types in 65 species.
• Bind negatively charged phospholipids in a calcium dependent manner.
• Composed of two major domains.
– Core region at the COOH terminal consisting of alpha helical disc with type 2
calcium binding sites. These interact with membrane phospholipids.
– Core domain (310 AAs) made up of 4 similar repeats (annexin repeats) 5 alpha
helices, 70 AAs long.
– Head region at NH2 terminal located on the concave side of the core region
which provides a binding site for cytoplasmic proteins. This may become
phosphorylated in some annexins and can cause affinity changes for calcium in
the core region or alter cytoplasmic protein interactions.

Annexin participate in:

• Vesicle transport
• Membrane organization
• Mitosis
• Inflammation reduction
• Apoptosis
1axn: Human Annexin III
Annexins: form structure to function , Gerke, V. & Moss, S, Physiol. Rev. 2002
 Glycolipid Transfer Protein
• Cytosolic protein that accelerates the intermembrane transfer of various
glycolipids.
• Catalyzes the transfer of various glycosphingolipids between membranes but does
not catalyze the transfer of phospholipids.
• May be involved in the intracellular translocation of glucosylceramides.
•  Has been found in brain, kidney, spleen, lung, cerebellum, liver and heart.

Glycosphingolipid binding
specificity is achieved through
recognition and anchoring of the
sugar-amide headgroup to the
GLTP recognition center by
hydrogen bond networks and
hydrophobic contacts, and
encapsulation of both lipid
chains, in a precisely oriented
manner within a 'molded-to-fit' 1swx
hydrophobic tunnel.

http://www.uniprot.org/uniprot/Q9NZD2
 Cytochrome C

• Cytochrome c is a component of the

electron transport chain in mitochondria.
• The heme group of cytochrome c accepts
electrons from the bc1 complex and
transfers electrons to the complex IV.
• Cytochrome c is also involved in initiation
of apoptosis. Upon release of cytochrome
c to the cytoplasm, the protein binds
apoptotic protease activating factor-1
(Apaf-1).
• Can catalyze several reactions such as
hydroxylation and aromatic oxidation, and
shows peroxidase activity by oxidation of
various electron donors.

Entrez Gene: Cytochrome C; http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/cytochrome-c.html