3 - Membranes

Benefits of membranes enabling the compartmentalisation of cells

is a functional barrier, so can separate reactions

eanbles formation of gradients across them (e.g. ATP synth, signal transduction)

Can organise and regulate enzyme activity

what parts of membranes can facilitate signal transduction

membrane bound receptors

lipids/proteins that can recruit and activate other proteins

how do membranes organise and regulate enzyme activities

they can bind enzymes, faciliating their localisation to specific compartments and membranes

purpose of phospholipase C

hydrolyses PIP2 → DAG (diacyl glycerol) and IP3.

both are second messengers. DAG remains in memb, IP3 rel into cytosol.

What signalling event activates phospholipase c

Gq-coupled GPCRs, where active Gq recruits PLC to memb, activating it.

Effect on intracellular calcium by phospholipase C activation

PLC cat PIP2 → DAG + IP3

IP3 binds IP3 receptors on ER/SR (Ca2+ channels)

cause opening and release of Ca into cytosol.

What does DAG activate

protein kinase C

Gram +ve vs Gram -ve

+ve have one cell memb

-ve have inner and outer

What lipid diversity in membranes is observed

chemical/structural diversity (type of lipid differs)

Lipid composition of individual membranes - differs between tissue, organelles, leaflets within same memb

(combo of FA chains can diff in lipids too)

what are the 3 types of lipid

glycerophospholipids

sphingolipids

sterols

what does the XX:Y nomenclature for lipids mean

XX= No of carbons in the chain

Y = no of double bonds

e.g. linoleic acid = 18:2, n-6 where n-6 is position of first double bond (count from methyl terminus (very end))

How can glycerophospholipids differ

lenght of FAs

double bond position and number

hydroxylation

cis- or trans- bonds in FA chains (cis gives 30 degree kink)

Examples of glycerophospholipids

phosphatidylethanolamine (zwitterionic)

Phosphatidylserine (anionic)

Phosphatidylcholine (zwitterionic)

phosphatidylinositol (anionic)

Cardiolipin (anionic)

why can PS and PE hydrogen bond to things

have a reactive amine group

why dont PI, PC, CL pack closely

they are large phospholipids

which organelle does the organelle-specific glycerophospholipid cardiolipin localise to

mitochrondria (inner memb) (mainly in the inner, matrix-facing, leaflet) (enzymes for its synth reside in this leaflet)

how are PIPs generated

by differential phosphorylation of the head group of PI

7 diff species

PI(4,5)P2 is most abundant in memb, PLC cat its conv to IP3 and DAG

interconversion regulated by kinases and phosphatases

Structure of sphingolipids

sphingoid base (most freq sphingosine), head group, N-acyl chain (which attaches to N of sphingoid base)

structure of sphingomyelin

sphingosine base, PC head, N acyl chain

sphingoid base vs a ceramide

ceramide is a sphingoid base with a fatty acyl chain

why are sphingolipids and cholesterol frequently found together in membranes

sphingolipids have a amide group that can interact with cholesterol OH group (H bond)

Do sphingolipids often have longer acyl chains than found in glycerophospholipids

yes

what are gangliosides a type of

glycosphingolipid - can have diff no and type of sugar bound to sphingoid base

Are glycerophospholipids, sphingolipids and sterols all phospholipids

no, only glycerophospholipids and sphingolipids are

structure of sterols

hydroxyl group and hydrocarbon tail e.g. cholesterol, (ergosterol in fungi and yeast) (sitosterol and stigmasterol in plants)

how does the presence of sterols in the membrane affect the membrane e.g. cholesterol

increases thickness & packing, reduces compressability.

Reduces the mobility of lipids and proteins in the memb.

what type of bond does cholesterol/sterols form with phospholipids in the membrane

hydrogen bonds

OH to polar head

interaction of cholesterol with sphingomyelin

has a high degree of complementarity

H bond formed between OH and SM

this ‘masks’ the OH of cholesterol

mediates the formation of lipid rafts

(both are major components of rafts)

what does lipid raft formation promote

clustering of proteins in/at the raft in the memb

how does cholesterol promote GPI-anchored protein recruitment to the raft

chol increases the order and stability of the memb, forming rafts

there is a preference for rafts due to the long/large GPI component of these proteins

Therefore clustering/interaction of these proteins is promoted.

is cholesterol essential for lipid raft formation

yes

what type of lipids have a preference for lipid rafts, important in signalling

glycosphingolipids

what is the bad type of cholesterol

low density lipoprotein (LDL)

builds up in arteries

LDL vs HDL

low protein:lipid ratio (low density of protein in their particles)

vs high protein:lipid ratio

why is HDL good cholesterol

removes cholesterol from the body to liver where it is broken down.

how does membrane lipid composition affect curvature

cylindrical lipids e.g. PC, PS form flat membranes

conical lipids form curved membs e.g. PE, PA (phosphatidic acid)

negative curvature vs positive curvature

neg - tail is widest bit (PE, PA)

pos- head is widest bit (PI)

which leaflet is PS mainly found in

inner leaflet

when on outside it signals for phagocytosis

what did the fluid mosaic model not account for

the wide variation in lipid/protein composition between membranes

but otherwise models the fluidity of membs and lateral mobility that lipids have well.

dynamic membrane domains/lipid rafts were a development of fluid mosaic model

can lipid rafts be protein-initiated or lipid mediated/assisted

both.

(e.g. actin clustering below pmemb can cause (protein) but other lipids may preferentially accumulate after this (lipid))

(e.g. GM1 form nanodomains driven by lipid-lipid interactions, they self aggregate).

why is lipid raft formation important for signalling

clustering of signalling receptors increases interaction and downstream signalling

e.g. thru cooperation, increasing rebinding probability

therefore can amplify signals

3 types of membrane proteins

peripheral

intergral

lipid anchored

peripheral vs integral memb protiens

peripheral dont span the memb like integral

peripheral associate with proteins or lipids in the membrane e.g. Ras associates with PS

how can membrane structure change to accomodate proteins

can change thickness (thicker or thinner), or the protein itself will tilt, to make sure the hydrophobic portion of the protein is not exposed to solvent.

what are the lipids around a memb protein known as

boundary or annular lipids, known as the footprint of the protein

bulk lipids = further from the protein, make up the rest of the membrane - their baviour does not change due to presence of the memb protein

what is the footprint region of a MP

unique environ of lipids around it, enriched in some, depleted in others

likely essential for function of MP

define nonannular lipids/lipid cofactors

lipids that bind region of MP within the memb e.g. pockets or between protein subunits

Req for MP function

how can molecular dynamics simulations useful for the study of MPs and membranes

can be used to predict the lipid environment of membrane proteins

see which ones interact with the MP (nonannular), and which ones form the footprint

specific lipids have preference for specific MPs

is the membrane traditionally observed in membrane protein structures

no, MPs are usually solubilised

‘reductionist’ model

why is preserving the lipid environment of MPs important for structural and fucntional studies

is a key functional part of the membrane proteins

influence structure too

as MPs evolved to be in the membrane, cannot simply just take them out.

what can solubilising MPs from the membrane cause

exposes hydrophobic regions

causes collapse/loss of integrity of the protein when removed

how is the Ryanodine receptor regulated by lipids

receptor more likely to be in open conformation when extracted at high lipid conc

how is the MscL protein regulated by lipids

it open and closes due to absence/presence of lipids in the central channel, protein conf stays relatively the same

how is the TRPY1 channel regulated by lipids

channel closed when PIP3 bound, open when it not bound. Allows Ca2+ thru

How are TRYC5 and TRPM8 regulated by lipids

lipid binds and inhibits receptor

so therefore regulates function

also seen with small molecule inhibitor Pico145

With new extraction methods, what was revealed about TRPC channels

also exists in a pentameric form in the memb, not just the tetrameric form previously seen

Therefore can see more native conformation/stoichiometry

pentameric may just be more unstable form

How may cholestrol promote dimerisation of some protiens

promotes close packing of membranes, increases bilayer thickness. Therefore MPs may change conf in response, and this conf may have greater affinity for other similar proteins

e.g. a tilted MP may be more vertical in cholestrol-rich memb, so can now interact more readily

could be why these proteins preferentially accumulate in thicker parts of membrane (e.g. rafts)

why would short TM domains of proteins promote aggregation

membrane has to distort/thin to accommodate the protein. Aggregating to similar proteins would reduce the distortion energy cost for each protein, so would be favourable

How may membrane density affect the opening and closing of channels

channels may req less energy to open if in a lower density membrane

so memb density could regulate opening

do cells actively remodel their membranes

yes

membranes differ from eachother

the same memb can differ over time e.g. diff points in the cell cycle

chemicals used to separate membrane proteins

detergents

amphipols

nanodiscs

peptidisc

SMA

how do detergents work

they mimic the lipid membrane by concealing Hphobic regions of proteins

are amphipathic, so Hphobic detergent and protien regions interact, and hphilic regions interact

allowing the MP to be solubilised from the memb

downsides of detergents

can be strong/harsh e.g. SDS (highly denaturing)

Even non-harsh ones can distort protein conf

can be costly

req a high conc of detergent to maintain micelles

(but are many options)

Some detergents more convenient for specific downstream analysis methods e.g. DPC for NMR (but they may not give the most native MP structure)

Can you get different results by using different detergents

yes, some denature more than others

Some give different stoichiometries of MPs

what are amphipols

amphipathic polymers

have high affinity for memb proteins

very good avidity (cause interaction is made up of many interactions)

benefits of amphipols

high avidity for memb proteins (many interactions formed with MP, so strong binding)

This gives it a very low Koff, so low Kd

so dont req maintain a high conc, like do with detergents

have a library of diff ones we can use

Now can be used to directly extract MPs from memb without use of detergent → more native conf

downsides of amphipols

are sensitive to Ca2+ and Mg2+ (form interactions with them instead of MP

lipid environ of MP not well maintained

benefits of nanodiscs

extract MPs whilst maintaining the native lipid environ

(traditionally tho they still ‘see’ the detergent for initial extraction, but hope they fold into nanodisc quickly)

Can dilute out detergent, therfore dont have any micelles present that could interfere with downstream analysis

Newer technologies (peptidiscs and SMA-based nanodiscs dont req detergents!)

downsides of nanodiscs

req right size disc tho (req it to fit, but too big and the MP can ‘drop out’

Disc can interfere with some analytical techniques

traditional ones req detergents so solubilise the MP from the memb, new ones (e.g. SMALPs or peptidiscs) dont req them (insert into memb and extract MP with native lipid environ)

(best case is if the protein has not ‘seen’ detergent)

time differs between extraction and entering nanodisc, so variation in how well protein is maintained in the movement

how do peptidiscs differ from nanodiscs

are short peptides that cover hPhobic regions of the protien. - self asseble around Hphobic regions - forming a stable water-sol particle

req detergents for initial extraction of MP, then add peptidisc, but can dilute out the detergent (as you can do with other methods)

are more ‘one-size-fits-all’ than nanodiscs, so are cheaper - can just add. Nanodiscs req use right size.

better for preserving

what are nanodiscs

amphipathic helices made of either membrane scaffold proteins (typically engineered from ApoA1 protein) or polymers (SMA) that encapsulates a small lipid bilayer - mimics cell memb

what is SMA

a polymer of repeating styrene and maleic acid

self assemble into nanodiscs

can extract MPs with native bilayer

but sensitive to divalent metal ions (-vely charged carboxylate groups bind to them) can cause aggregate and precipitate

Some proteins req metal ions for function/conf, so is a big hinderance for study of some

Modified SMA polymers can improve tolerance to metal ions

how can the native lipids surrounding a MP in e.g. SMA-derived nanodiscs or peptidiscs be identified

Mass spectrometry

Downsides of extraction of membrane proteins by all detergents, amphipols, nanodiscs, peptidiscs

Dont allow study of MP in a closed system

therefore cant study effect of membrane potential on structure or function (some dependent on memb pot, e.g. voltage-gated ion channels)

benefits of vesicles/liposomes for MP study

allows for a closed system to assess the MP

can do buffer exhange to change the membrane potential

are more native bilayers than e.g. nanodiscs

can tailor vesicle size and lipid composition

downsides of vesicles/liposomes for MP study

likely req tailor vesicle size and composition

may have higher memb curvature than native bilayer, so could perturb protein conformation and function

Protein orientation can affect downstream analysis

multilamellar structures (stacks of bilayers) can also form - affect downstream analysis

can degrade easily - short timeframe to perform experiments following extraction

How can the stability of liposomes be improved

can create hybrids, using polymers

helps maintain stability, so therefore protein stability/activity for longer

(months not days)

problems with overexpressing MPs for extraction

Not increasing lipid formation with it, so get accumulation of MP in memb, higher concs are not native.

may get aberrant structures - native conf dependent on key lipids, may not be enough of them

or misfolding as potentially overloading synthesis machinery - so yield of functional MPs may not be any higher

may need to humanise expression hosts e.g. use Yeast, not bacteria

why is it important to study membrane proteins

lot of therapeutics target MPs, so aids effective development

why it is also useful to know more about key lipids involved in their regulation, can target these regulatory interactions/sites

gram -ve vs gram +ve bacteria

+ve have no outer memb

just a thicker peptidoglycan layer

what structure do most MPs in gram -ve bacteria have

beta barrel

the PMF in gram -ve bact occurs across inner memb, with no ATP in periplasmic space. How may outer MPs be regulated

interact with inner memb proteins

interact with periplasmic chaperones

how are alpha helices and beta strands often found in the memb

alpha helices associated laterally

beta strands form sheets that often form cylindrical structures e.g. b barrel

MPs have diff evolutionary constraints than soluble proteins. What interactions do MPs have to form for hphilic regions to be happy within a hphobic membrane

H bond formation with the backbone is needed to satisfy polar groups (the carbonyl and NH groups), as these would otherise prefer to be buried in a Hphobic core, or face solution (which is what soluble proteins do)

Most residue side chains that face the acyl chains of the bilayer must be hphobic

how do helices and beta strands satisfy their lipid-facing hydrophilic regions

a helices form h bonds within the backbone, so can be inserted by themselves into the membrane - then can associate laterally

beta strands have h bonds within their backbone but also between strands and form sheets - therefore these otherwise polar groups would not like facing lipid

Strands could not be inserted on their own (energetic cost would be too high)

Hbonding of beta sheets can therefore occur between residues distant in the aa sequence.

what are OMPs

beta barrel outer membrane proteins

form pores in gram -ve bact outer memb, mito and chloro

structure of OMPs

9-11 residues in as strand, stands are on a tilt in the memb

even no of antiparallel strands form a barrel

their n and c termini are in the periplasm

variation in loops observed more than strands obvs

Hbonding is the strongest stabilising interaction holding them together

what is the aromatic girdle

ring of mostly aromatic residues (~40%) found at each end of beta barrel proteins, rest are often hphobic still

one half of a girdle is hphobic (face acyl chains of lipids), other is hphilic (solvent facing) (interact with heads of lipids)

e.g. tyrosine and tryptophan have non-polar R groups that face hpobic lipid region, polar amide bonds that face solvent

they stabilise the interface between protein and lipid mols

why are OMPs hard to solubilise

smallest ones have 8 TM b-strands, so lot of hphobic residues that favour facing lipid

do hphobic and hphilic residues alternate in beta strands

yes

which residues are polar in OMPs

solvent facing ones, either at barrel ends, or in facing the interior e.g. channel

what is hard to capture of native membranes in artificial membs

lipid diversity, other proteins, asymmetric distribution of lipids between leaflets

do detergents form bilayers

no, form micelles

why are experimental conditions limited for studying MPs

req maintain the membrane

so req try to accommodate for protein and the memb

difficulties of working with MPs

hard to produce in large quantity

hard to unfold and solubilise completely

often complex

folding/unfolding often not fully reversible

how to study MP folding/unfolding

extract MP, denature in high [urea]

add detergent or lipid

can dilute out urea, get refolding into micelle/bilayer

then can repeatedly change [urea] to observe unfold/refold

how easy it unfolds informs on MP stability

what is circular dichroism

measure the differential absorption of left and righ-handed circular polarised light by the sample. Perform it across a range of wavelengths.

secondary structures have distinct fingerprints (specific minima and maxima)

Can use software to estimate quantitiy of each structure

So can be used to measure unfolding as plot changes/the quantity of each

How can cold SDS page be used to monitor unfolding

migration rate (therefore distance) of folded/unfolded protein differs, so can see proportion of unfolded/folded by seeing their relative insensities.

So can measure it over time in e.g. a denaturing condition

How can tryptophan environment be used to monitor unfolding

measure fluorescent intensity over range of wavelengths

As microenviron of tryptophan residues change in protein the fluorescent intensity at a given wavelenght changes

unfolded and folded state have diff intensity-wavelength plots, can see shift of curve as unfold/fold more

possible peak flattening, sharpening, shifting in a direction, all depends on specific protein

what does a larger more negative delta G mean for a folding reaciton

folding is highly favourable, protein is highly stable