Macro membrane proteins Flashcards
lehniner 11 biological membranes & transport
what are the structural properties of biological membranes
fluid mosaic model
fatty acyl chains in membrane interior form fluid hydrophobic region
integral proteins float in this sea of lipid held by the hydrophobic effect
protein and lipids are free to move laterally in the plane of the bilayer
but movement of either from one face to the other is restricted
high unsaturated fat - melting temp = lower
how are membranes specialized for a particular function
peripheral - on surface - attached to phospholipid hydrophilic part of lipid i.e. cytocrome C
integral - single transmembrane helix - bitf of a domain inside and outside
peripheral protein - covalently attached to lipid
glycolipid - outside cell
glycoprotein
what are the structural features of membrane proteins
hard to crystallise
insoluble in buffers an denature in oragnic solvents
if u add detergents to the aqueous buffer - stabilize the proteins = protein-detergent omplexes are the starting material for crystalization
reluctant to yield crystals that diffract x rays to high resolution
the active and passive transport of solutes across biological membranes
simple
non polar compounds
down conc gradient
facilitated diffusion from outside to inside = down electrochemical gradient
primary active transport i.e. the ABC transporters and SERCA transporters - against electrochemical gradient by coupling to the energy released from ATP hydrolysis
outside is transferred inside with the hydrolysis of ATP
the energy released by ATP hydrolysis drives solute movement against an electrochemical gradient
in secondary active transport -
against electrochemical gradient
driven by ion moving down its gradient
a gradient of ion X often soim has been established by primary active transport
movement of down its electrochemical gradient now provides the enrgy to drive cotransport of a second solute (S) againt its electrochemical gradient
ion channel
down electrochemical gradient
may be gated by a ligand or ion
ionophore mediated ion transport down electrochemical gradient but ion is encased in a carrier
integral membrane topology
how can we tell how many times a helical protein traverses the membrane
a sequence of 20-25 residues is just long enough to span thickness of 30A of the bilayer 1.5A per residue in an a helix
we calculate the hydropathy index from the non polarity of sequence of amino acids using 20 residue windows = shows transmembrane helices
side chains Tyr an Trp serve as membrane anchors
they can interact simultaneously with the central lipid phase and with the aqueous phases
residues of tyr and trp are found predominantly where the non-polar region of acyl chains meets the polar head group region
charged residues are found in aqueous places or within protein pores
potassium channel
beta barrel membrane proteins
20 or more transmembrane segments form beta sheets that line a cylinder
every second residue is hydrophobic
alternating one pokes up the the other down
the hydropathy plot is useless in predicting transmembrane segments for proteins with beta barrell tifs but comparison with known beta barrell does work
the electrochemicl chemical gradient
when two aqueous compartments containing unequal concentrations of a soluble compound or ion are separated by a permeable membrane
moves across by simple diffusion till the 2 compartments have equal concentrations
when ions of opposite charge are separated by a permeable membrane ther is a transmembrane electrical gradient - membrane potential (Vm)
the direction in which a charges slute tends to move spotaneously across a emmebrane depnds on both the chemical gradient and the membrane potential. = electrochemical gradient/potential
energetics of membrane protein transport
to pass through a lipid bilayer a polar solute must first lose its solvation shell and then diffused about 20 A through the lipid which it is poorly soluble in
activation energy - delta g energy reqired is high but is reained as the polar compound leaves the membrane on the otherside and is rehyrated
intermediate stage of transmembrane passage is a high energy state comparable to the transition state in an enzyme catalysed reaction
delta g is so large that pure bilayers are virtually impermeable to polar solutes
membrane proteins lower the activation energy for transport by providing an alternative path through the bilayer for specific solutes
3 general classes of transport sys
transporters differ in the no of solutes transported and the direction in which each is transported
channels facilitate passive diffusion down an electrochemical gradient
gated or ligan
Uniport - 1 in 1 direction
cotransport:
symport - 2 in same direction @ same time
antiport - one ion in and one out @ same time
regaress of energy requiring or no
ion selective channels move ion-organic ions across membranes
together with ion pumps sodium potassium ATPase
they determine plasma membrane permeability to specific ions and regulate the cytoslic [ion] and membrane potential
ion channels have three distinctive differences from pumps
- the rate of flux is near the theoretical limit for ion diffusion (10 to the power of 7 ions)
- they are not saturable - rtes do not approach a maximum at high [ion]
- they are gated - open or closed in response to some cellular event either binding of a ligand or change of transmembrane potential (Vm)
the bacterial potassium channel
related to the volate gated potassim channel of neurones]]]most similar in the pore region - containing the selectivity filter - allow potassium to pass 10k times faster than sodium
consists of 4 identical subunits that span the membrane and form a cone that surrounds the ion channel with the wide end facing the extracellular space
each subunit
2 transmembrane helices as well as a 3rd shorter helix that contributes to the pore region
outer cone is formed by 1 of the transmembrane helices of each subunit
entryways to the channels have -vey charged residues which attract and increase the local conc of cation sodium. + potassium
potassium is hydrated in the wide waterfiled channel (inner surface)
electric dipoles on the short a helices help to draw potassium into the channel
halfway through the membrane the channel narrows in the region of the selectivity filter forcing the k+ ion to shed it hydration shell
backbone carbonyl oxygen atoms from each of the subunits replace the water forming 4 coordination shells through which the potassium ions move
not possibe for sodium as it is too small to contact all the potential carbonyl group ligands - this is the basis for slectivity of potassium over sodium
of the 4 potetial potasssium binding sites only 2 are occupied in the crystal structure the other 2 are occupied by water
movement of potassium is concerted drawing a new potassium ion into the channel by the short alpha helices pushed the potassium ions in the selctivity filter through the pore
open and shut states
bacterial potassium channels are connected to gating gomains which open and shit the channel based on different signals, such as voltage or the binding of a ligand
a comparison of the open an closed chanel structures shows that the gating dmains work by twisting the 4 subunits of the channel
the crystal structure of the closed channel has several potassium ions in the channel
aquaporins
provide channels for appid movement of water across plasma membrane
erthryocytes have a high [AQPs] to allow them to swell and shrink due to changes in osmolarity upon passing through the renal medulla
proximal renal tubula cells of the kidney reabsorb eater uring urine formation t
they have 5 diff AQPs in theire plasmam memebrane
high rate of flux suggests wwater molecules move through as a continuous stream in the direction of the osmotic gradient
but a continuous stream of water would allow the passage of protons as this would collapse a membranes electrochemical potential
water interacts with protons - hydroxanium ions = passage of protons are drawn = collapse of membrane electrochemical potential
how do allow molecules to pass through the channel but not water
APC tetramer structure
each monomer forming a transmembrane pore of diameter sufficient to allow passage of water in signle file
each monomer consists of 6 transmembrane helices and 2 shorter helices each contains Asn-Pro-Ala (NPA)
short NPA contains helices extend towards the middle of the bilayer
their NPA regions overlap in the middle of the membrane to form part of the selectivity filter
+ the strcture that allows only water to pass
the residues lining the channel of each AQP-1 monomer are genrlly non polar
the 2 Asn residues in the NPA looops and backbone carbonyl oxygens form hydrogen bonds with individual water as they pass through
generally hydrophobic environmnt
aquaporins dont leak hydrogen ions
critical ARg and His residues and electric dipoles formed by the short helices of the NPA loops provide positive charegs in key positions
these positive charges repel any H2o that might otherwise leak through
primary active transport i.e. the ABC transporters and SERCA transporters - against electrochemical gradient by coupling to the energy released from ATP hydrolysis
ABC transporterS
ATP binding cassette transporters pumping amino acids, peptides,
proteins, metal ions, various lipids, bio salts, many non polar compounds, including drugs out of cells against concentration gradient
drug resistance
MDR1 in humans = resposible for the resistance of certain tumours to some otherwise effective antitumour drugs
broad substrate specificity for hydrophobic compounds all anticancer drugs
by pumping these drugs out of the cell MDR1 prevents their accumulation within a tumour and blocks their therapeutic effects
MDR1 integral membrane protein wih 12 transmembrne segments and 2 ATP binding cassettes
All ABC transporter have 2 neuceotide binding domains )- motor that can be coupled to ifferent pumps/channels) and 2 transmembrane domains
most ABC transporters act as pumps or ion channels that are switched on or off by ATP hydrolysis
CFTR - cystic fibrosis transmembrane conductance recptor
disease causing mutation occurs in the ABC cassettes in one of the nucleotide binding domains
25% energy of the cell goes to = sodium potassium ATPase - memebrane potential -70mV
out 3 sodiums
calcium must be pumped out of the cytoplasminto endoplasmic reticulm
lower than in blood plasma
if it were higher it would combine with inorganic phsophate (high because of aTp hydrolysis) to form insoluble calcium phosphate
calcium ions are Pumped out of the cytosol bythe plasma membrane calcium pump or into the lumen of the endoplasmic
Reticulum or sarcoplasmic reticulum - is the surrounding of the muscle fibre protein.
the passage of calcium into the muscle cells = initiates muscle contraction then we have to pump calcium’s to get things back to normal.
sarcoplasmic and endoplasmic reticulum calcium pump SERCA:
exists in 2 forms
phsphorylated
favours a conformation with a high affinity calcoium binding site exposed on the cytoplasmic side
dephosphorylatin favours one with a low calcium ion affinity on the lumenal side
energy from aTP hydrolysis during one phos-dephos cycle drives calcium ions across the membrane against a large electrochemical gradient
phosphorylation siganl si transmitted by a conformational change that alters the calcium ion affinity and opens a path for calcium ion release on the lumenal side
during one cycle the N domain tips about 20 degrees brining the ATP binding site close to the phosphosrylated ASP351 and the A domain twists by 90 degrees
seconadry active transporters
solutes move against their electrochemical gradients by coupling to an ion moving down its own gradient i.e. lactose transporter of e celi
in baceria primary transport of hydrogen ions out of the cell driven by oxidation of a variety of fuels establishes both an hydrogen ion graient and an electrical potential
secondary active transport of lactose into the cell involves symport with hydrogen ions
uptake of lactose byt the lactose transporter against its concentration gradient is entirely dependent on this inflow of hydrogen ion driven by the electrochemical graient
when the energy-yielding metabolism is blocked by cyanide the lactose transporter only equilibrates the [lactose] via passive transport
passive transporter unless its linked to the proton transporter through hydrogen concentration
the rocking banana model
rocking motion between the 2 domains driven by substrae binding and proton movement
alternately eposing the substrate binding domain to the cytoplasm and to the periplasm
mutations affect glu325 and arg302 have the same effect on cyanide = rocking banana probably driven by their titration
