membrane proteins

Question 1

describe structure of phospholipids and lipid bilayers

Answer 1

2 lipid tails, one polar phosphate head

since it has both hydrophillic and hydrophobic attributes it is called ampipathic

bilayer sheets form spontaneously in aqueous environment, non covalent forces in membrane formation

if there is 1 lipid tail instead of 2 it forms a detergent micelle instead of lipid bilayer

Question 2

describe properties of membrane and its proteins

Answer 2

membrane is 6-10 nm thick

contains mostly lipids and membrane proteins with carbohydrate attached to both

protein: lipid ratio varies from 1:4 (myelin) to 4:1 (mitochondrial inner membrane)

lipid acts as a solvent for membrane proteins

membrane proteisn move around freely within the lipid bilayer, within the plane of the lipid bilayer

2 sides of membrane are asymmetric with different properties

membrane components diffuse rapidly latterally (across their layer) but do not, or very slowly diffuse across layers

Question 3

whar are lipid rafts

Answer 3

lateral separation of lipids to produce areas that are relatively organised in an otherwise highly fluid disorganised bilayer (in the membrane)

lipid rafts are high in sphingolipids and cholesterol, show detergent resistance and increase the thickness of bilayers

Question 4

what is difference between water soluble and membrane globular proteins

Answer 4

water soluble diffuse freely in 3D

in membrane 30-90% freely diffuse in 2D space but not from one side to another

water soluble have a core buried of hydrophobic residues with hydrophillic residues on the outside, membrane proteins have large or small areas of surface hydrophobic residues

some membrane proteins have hydrophillic centres and hydrophobic surfaces

Question 5

describe the membrane of bacteria

Answer 5

bacteria (gram negative) have a double membrane (double bilayer) with thickness of 25nm

carbohydrates are attached to surface of outer layer, inner layer contains transport proteins etc

space between layers is called periplasmic space, roughly 12nm thick

Question 6

what is spectrin

Answer 6

fibrous membrane protein found in erythrocytes and stablises the shpae of the cell, is membrane bound/ anchored but exists intracellularly

Question 7

what are examples of globular membrane protein functions

Answer 7

transport, electrical activity, signal transduction, energy conversion, adhesion between cells

Question 8

what is role of fibrous membrane proteins

Answer 8

usually have structural roles

Question 9

what are different types of membrane proteins how do they interact w membrane

Answer 9

integral membrane proteins span the whole membrane/ transmembrane proteins

peripheral proteins either bind to integral proteins or form links with phosphate heads by electrostatic attractions or H bond interactions

lipid linked membrane proteins bind to lipid bilayer by means of one of 3 lipid tails, either a GPI anchor, isoprenes or fatty acid acylated

integral membrane proteins bind using hydrophobic interactions, these are amphipathic, surface regions outisde membrane coexist with water, so these are hydrophillic with polar sidechains, carbohydrate if present is on outside surface only, surface regions inside the bilayer face environment with no H binding capacity, common AAs in the lipid part are val, ile, leu

Question 10

how are different types of membrane proteins eluted (removed)

Answer 10

peripheral proteins are water soluble, can be purified, these can be eluted (removed) by chromatography, use of salt, pH changes or chelators

integral membrane proteins are insoluble in water in absence of detergent, elute these by chromotography only by destroying the lipid bilayer with organic solvents or detergents

Question 11

how are integral membrane proteisn solubilised

Answer 11

use of gentle, non-ionic detergents, the cmc (critical micelle concentration) is the concentration above whihc the detergent can form micelles

higher cmc is better for solubilising membrane proteins

Question 12

give examples of detergents used in membrane protein solubilisation

Answer 12

SDS (sodium dodecyl sulphate) is charged and denatures proteins, effective for SDS-PAGE

triton X-100 is a neutral detergent, hydrophobic part is p-t-octyphenol, hydrophillic part is polyoxyethylene repeating unit; CH2CH2O, there are 9-10 repeating units, has a low cmc of 0.3 mM

octyl-beta-D-glucoside is another neutral detergent used for protein solubilisation, hydrophillic part is glucose, hydrophobic part is octanol, high cmc of 23mM, more effective for solubilisation

Question 13

what is required to purify a membrane protein

Answer 13

use of detergent to break up the bilayer

protein is contained in micelles which are purified, addition of phospholipids with detergent creates a phospholipid vesicle with the protein in which can be extracted

Question 14

descibe secondary structure of membrane proteins

Answer 14

alpha helices can pass through inner part of the lipid membrane, beta strands cannot pass through lipid membrane since H bonds on outside are not hydrophobic, however beta barrels can exist

no other secondary structures can exist

alpha helical proteins have predictable secondary structures, beta sheet ones do not

to pass through membrane an alpha helix of 20 AAs is required

either all alpha helical or all beta sheets, 3 groups of structures;

monotropic membraen proteins (are attached to only one side of membrane and do not spand the whole way)

transmembrane alpha helix

transmembrane beta barrel

Question 15

how is secondary structure of membrane proteins predicted

Answer 15

secondary structure predictions based on methods for water soluble globular proteins do not work for membrane proteins, from the sequence it can be accurately predicted whether a protein will be water soluble or membrane bound using a different method called hydrophobicity plots

Question 16

describe hydrophobicity plots

Answer 16

a numerical scale of hydrophobicities for the 20 amino acids is needed, these values are based on the free energy transfer of AA sidechains from aqueous to hydrophobic pase and on the observed distribution of sidechains between the surface and interior proteins of known structure, a negative value means the AA is hydrophillic, a positive means it is hydrophobic

to create a profile assign a hydrophobicity score to each amino acid residue in the sequence, slide a 21-residue window along the protein sequence, calculate the average hydrophobicity inside the window, plot the average vs the position of the window in the sequence

positive peaks indicate predicted membrane spanning helices, negative valleys indicate hydrophillic regions

this method assumes that membrane spanning alpha helices are hydrophobic

porin is a membraen protein with beta sheet structure, has not long hydrophobic sequences

this method only works for alpha helices

Question 17

what are mechanisms of GPCRs

Answer 17

large displacement in helix 6 and an opening of a deep hydrophobic cleft on the intracellular side of the receptor which allows G protein to associate

activated G protein breaks apart, free alpha subunit triggers chain of reactions that alters cell metabolism

new G protein binds, the receptor can activate hundreds of G proteins before ligand detaches

Question 18

describe the photosynthetic reaction centre

Answer 18

photosynthetic reaction centre is an example of a transmembrane alpha helical protein

subunits are : C type cytochrome on the extracellular side of the membrane, a peripheral protein, L subunit containing 5 membraen helices, M subunit containing 5 membraen helices and the H subunit which contains 1 membrane helix

cyctochrome contains prosthetic groups which pass down “light energy” through the protein, absorbed photons produce the energy needed to donate electrons to an acceptor

these electrons are transmitted from chlorophylls to other groups (quinones), the quinone is then released for further action

Question 19

what is porin

Answer 19

porin is an example of a beta sheet protein, it is abundant in bacteria, also found in chloroplasts and mitochondrial membranes, it has no predicted long stretched of hydrophobic residues

it permits the uptake and disposal of small molecule nutrients and waste which have a mol weight of less than 600

Question 20

describe the structure of porin

Answer 20

composed of 3 beta barrel monomers

each monomer is a 16 stranded beta barrel in which the strands are antiparallel and 7-16 residues long

hydrophobic and hydrophillic residues alternate along the beta strands, giving rise to hydrophobic outer surface and hydrophillic inner surface

large molecules cannot enter due to the size of the water filled channel centre which is only about 0.9nm long and 0.8nm in diameter

channel also has one side lined with positive AAs (his, lys, arg) and the other with negative (glu,asp) which effects what passes through

Question 21

what are the types of transporters

Answer 21

3 types:

active pumps which use ATP, they can pump 1-1,000 ions/s

channel proteins, they can transport 10^7-10^8 ions/s

transporters, which can transport 100-10,000 molecules/s

Question 22

describe basic structure of human sodium/ potassium channels

Answer 22

they are tetrameric

each subunit contains 6 alpha helices, S1-S6

there is a H5 loop between S5 and S6 and a N terminal segment that activates/inactivates the open channel

Question 23

describe structure of human potassium channel

Answer 23

the main chain C=O groups in potassium channel tht point into the pore constitute binding sites for 3 potassium ions

potassium ions are dehydrated and this hydration is compensated for by the C=O groups

core helices point towards potassium ions to neutralise their charge via the helix dipole effect

repulsion between successive potassium ions leads to their movement

C=O groups are part of covalent mainchain and so do not move

channel protein is 100 times more specific for potassium over sodium due to different ion sizes and hydration levels

sodium ions are much smaller and have a higher solvation energy

Question 24

why are erythrocytes robust

Answer 24

they need to be robust due to squeezing through capillaries

they have lifespan of 120 days, this is achieved with help of many contacts made with the cytoskeleton (peripheral membrane proteins), unlike other cells

Question 25

how is spectrin purified

Answer 25

spectrin has mol weight 250,000-260,000

erythrocytes are broken down by lysis, causing loss of Hb, leaving the “ghost”

the ghost is resealed, detergent is used to remove lipids and integral proteins

a very low ionic strength salt solution is used to remove some peripheral proteins

the shell is left which contains a network of peripheral proteins including spectrin

Question 26

describe structure/function of spectrin

Answer 26

spectrin connects with ankyrin in the bilayer, it also connects to short actin filaments with the help of band 4.1 protein, a polygonal network of this is created to form the cytoskeleton of the erythrocyte

spectrin has a repeat of 106 AAs that forms a triple stranded alpha helix with 18 or 20 repeats total

Question 27

what are pore forming colicins

Answer 27

are a family of 3 domain toxins (600AAs) produced by E coli and related bacteria

they form ion channels in the inner membrane of target cellls

colicin has 3 domains; A,B and I that form a Y shaped structure

B is a receptor which binds to the external membrane

A is used for translocation to the periplasmic space

I is used for ion channel formation