1. Lecture 07 (Chapter 12)
Membrane Proteins

2. 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);

3. Membrane Protein Functions
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.

4. 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

5. Fluid Mosaic Model of Lipid Bilayer

6. 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

7. 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.

8. 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).

9. 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.

10. 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

11. 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

12. 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)

13. 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 selectively (by a factor of 10, 000 over Na+, despite the sub-angstrom difference in ionic radii).
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).

14. 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.

15. Calcium-activated potassium channels
Based on conductance (big, intermediate, and small), can be sub-divided into:
BK channels
IK channels
SK channels

16. 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

17. 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.

18. 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 [Ca2+] 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;

19. 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-spanning alpha helices (M1 and M2).
1p7b
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;

20. 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

21. 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.
Voltage-gated potassium channels as therapeutic targets, Wulff et al., 2009

22. 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;

23. Calcium channels - Overview
Two types of ion channels with selective permeability to calcium ions
Voltage gated calcium channels
L-type (High voltage activated)
Found in muscle, bone, ventricular myocytes
P-type (High voltage activated)
Found in Purkinje neurons in cerebellum
N-type (Hight voltage activated)
Found in brain and peripheral nervous system
R-type (Intermediate voltage activated)
Found in cerebellar granule cells, other neurons
T-type (Low voltage activated)
Found in neural cells, bone
Ligand gated calcium channels
IP3 receptor
Ryanodine receptor
Two pore channels
Cation channels of sperm
Store operated channels
Ion channels: A paddle in oil, Lee, 2006;

24. 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

25. 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: ].
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

26. 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
memory
IP3R represents a dominant second messenger leading to the release of Ca2+ into the cytosol from intracellular store sites (i.e., SR lumen).
IP3 binding core (IBC) of IP3 receptor with bound IP3 molecule
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;

27. 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.

28. 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.

29. 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:
By amphipathic α-helix parallel to membrane
By a hydrophobic loop
By a covalently bound membrane lipid (lipidation)
By electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion)

30. 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)

31. 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 -

32. 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.

33. 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

34. 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' hydrophobic tunnel.
1swx

35. 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;