Membrane proteins

what % of proteiome proteins are membrane associated

20%

give a pharmacological example of membrane proteins importance

B2AR regulate muscle relaxation, agonists of B2AR (membrane protein) are used in treating asthma

what are 6 common roles of membrane proteins

markers, enzymes, structural, channels, transporters, receptors

how does a membrane protein act as a receptor?

the protein receives a signal and sets in a play of events ending up with a response to signal

example of how a membrane protein acts as a receptor?

rhodospin, light activated involved in visual perception, many bacteria use it to harvest light energy to generate ATP

how does a membrane protein act as a transporter?

regulates the transport of chemicals from one side of a biological membrane to another e.g insulin receptor

can be one cell to/from cyctoplasm or cell-to-cell via gap junctions

how does a membrane protein act as an ion channel?

opens up a continuous pore through the membrane, through which ions flow according to their concentration gradient

how do membrane proteins have structural importance?

provide mesh-like support inside cell membrane e.g ankyrin, spectrin, tropomyosin

example of membrane protein that acts as an enzyme

those used in ETC

what are the 3 structure types of membrane proteins

integral membrane spanning, peripheral membrane attached, anchored

what are integral membrane spanning membrane proteins?

permanently embedded within the lipid bilayer of the cell membran, have hydrophobic regions that interact with the lipid bilayer, and they can span the entire membrane (transmembrane proteins) or be partially embedded

what are peripheral membrane attached membrane proteins?

temporarily associated with the membrane surface, either through interactions with integral membrane proteins or with the polar regions of the lipid bilayer. they do not span the membrane, and they can be easily removed from the membrane without disrupting its integrity.

what are anchored membrane proteins?

attached to the membrane via covalent bonds to lipids embedded in the membrane.They are not directly embedded in the lipid bilayer, but their lipid anchor allows them to associate with the membrane.

example of membrane protein as a marker - infection

1. infected cell digests foreign protein into proteome

2. resulting peptides transported into ER and located onto MHC

3. MHC with peptide is traffiked to cell surface and ‘marks’ surface of infected cell

4. cytotoxic T lymphocyte (CTL) recognises flag and engulfs infected cell

how can peripheral membrane proteins be removed

using high salt buffers as they’re attachedwhen to membrane only by electrostatic attractions

in what membrane proteins are the core hydrophylic

ion channels as ions are water soluble molecules

how many aa residues are present in a-helices core membrane proteins

25, side chains on outside of every helix

how many aa residues are present in b-sheets core membrane proteins

9-11 residues, side chains point alternative in and outside of barrel

what stabalises b-barrel porins?

trimeric interactions

what do trimeric interactions do

enhances stability and regulate transport of b-barrel porins using b-strand interdigitation, hydrophobic core packing, and loop-loop contacts

what are b-barrels

non-specific porins, substrate flux down diffusion gradient, hydrophylic core to allow water soluble molecules to pass, stabalised by trimeric interactions

what determines the likelyhood of aa’s being in membrane spanning reigon?

octanol: water positioning of aa

statistical observation of occurance in kown membrane spanning reigons

what is the main steps in studying membrane protein strucutre and function

1. expression

2. isolation

3. solubalisation

4. purification

5. reconstruction

give 2 examples of natural sources used to get expressed protein in membrane protein analysis

protein bacteriorhodopsin from bacteria halobacterium halobium in salt flats

protein acetylcholine receptor from torpedo (electric ray) electric organ

what are the benefits for using prokaryotic systems for expression in analysis of membrane proteins

low cost, simple culture conditions

what are the disadvantages for using prokaryotic systems for expression in analysis of membrane proteins

low yeild, modifications occur, solubility problems due to improper folding and formation of inclusion bodies

what are yeast, virus infected insect cells, and mamalian cells used for in membrane protein expression in analysis

used for eukaryotic proteins only

what are cell free expression systems used for in membrane protein analysis

toxic proteins, or those with incorporation of unnatural aa’s or labels

why are detergents necessary for purification of membrane proteins in analysis

membrane proteins are not water soluble so they must be solubalised prior to purification

what are the three classes of detergents

ionic, non-ionic and zwitterionic

what are ionic detergents

harsh, can disrupt hydrophobic and ionic forces and disrupt protein folding

e.g cholic acid and SDS

what are cholic acid and SDS examples of

ionic detergents

what is the ‘best’ type of detergent

non-ionic detergents

what are examples of non-ionic detergents

Triton-X-100, maltosides, tween series, NIG-CHAP

what are non-ionic detergents

less harsh, disrupt hydrophobic forces holding protein in membrane only

eng Triton-X-100, maltosides, tween series, NIG-CHAP

what are zwitterionic detergents

no net charge, can distrupt protein-protein and protein-lipid interactions

e.g CHAPS, sulfobetaines

what are CHAPS, sulfobetaines examples of

zwitterionic detergents

how do detergents distrupt the bilayer

they integrate their single hydrophobic tail into membrane, destabalising the bilayer structure and increasing solubility

what must the conc of detergent reach so it can form micelle around proteins in membrane?

critical micelle concentration

what is most common problem of using detergents and solution

solubalisation of membrane proteins can lead to loss of activity and denaturation, can be overcome by finding detergent that has correct length ensuring proper mimicking of lipid environment

what are 3 common methods of purification of membrane proteins

ion exchange chromatography, gel filtration and affinity chromatography

all need CMC throughout

what are 4 methods of reconstruction of membrane proteins in membrane

dialysis and dilution

gel filtration (size exclusion)

adsorption to polystyrene beads

precipitation

when is dialysis and dilution used in membrane protein reconstruction

detergents with high CMC as don’t need to remove a high % of detergent to trigger micelle.

detergent monomers pass through porous tube, proteins and lipid forms membrane

when is gel filtration used in membrane protein reconstruction

good for detergents where micelle is much smaller than lipid embedded in protein, gets rid of single detergent molecules

when is adsorption to polystyrene beads used in membrane protein reconstruction

good all round, lipids don’t interact with polystyrene beads whereas most detergents do

example of precipitation for reconstruction of membrane proteins

SDS + K+ → KDS (insoluble)

what is high and low resolution stats

high = 1-2 A

low = 2-4 A

how does EM generate 3D data?

tilting specamin in the electron beam allowing reconstruction of 3D model

what material is analysed in EM?

solutions of proteins ~250 kDa

2D crystals

what material is analysed in X-ray crystalography?

3D crystals

what material is analysed in NMR?

small proteins <30 kDa

which method of viewing proteins is dynamic

NMR

what is structure of aquaporins

hourglass shape

6 predicted TM helices - predicted 2-fold internal repeats with same topology

2 conserved loops not in membrane

what is the problem of water transport through membranes

protons move through water by hopping from one H2O to another using hydrogen bonds, solution is to break chain of H-bonds or disrupt alignment of H2O molecules

how do aquaporins transport water without proton leakage

1. at entrance H2O forms H-bond with C=O groups and neighbouring H2O

2. in centre of pore H2O is H bonded to Asparagine Asp residues

3. dipole flip mechanism - H2O enters pore aligned in one direction but at centre they flip their dipoles

4. this reorientation interrupts H-bonding network, preventing proton transport

what are the 2 subfamilies of aquaporins

water only carriers

water + glycerol carriers

what aquaporins are found in secretory glands

AQP 5 in luminal side allowing water to pass into sweat, tears and saliva

what aquaporins are found in lungs

AQP’s 1, 3, 4 and 5 in various places in nasophaynx, airways and alveoli