membrane proteins Flashcards
describe structure of phospholipids and lipid bilayers
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
describe properties of membrane and its proteins
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
whar are lipid rafts
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
what is difference between water soluble and membrane globular proteins
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
describe the membrane of bacteria
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
what is spectrin
fibrous membrane protein found in erythrocytes and stablises the shpae of the cell, is membrane bound/ anchored but exists intracellularly
what are examples of globular membrane protein functions
transport, electrical activity, signal transduction, energy conversion, adhesion between cells
what is role of fibrous membrane proteins
usually have structural roles
what are different types of membrane proteins how do they interact w membrane
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
how are different types of membrane proteins eluted (removed)
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
how are integral membrane proteisn solubilised
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
give examples of detergents used in membrane protein solubilisation
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
what is required to purify a membrane protein
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
descibe secondary structure of membrane proteins
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
how is secondary structure of membrane proteins predicted
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
describe hydrophobicity plots
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
what are mechanisms of GPCRs
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
describe the photosynthetic reaction centre
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
what is porin
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
describe the structure of porin
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
what are the types of transporters
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
describe basic structure of human sodium/ potassium channels
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
describe structure of human potassium channel
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
why are erythrocytes robust
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
how is spectrin purified
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
describe structure/function of spectrin
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
what are pore forming colicins
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
