Membrane Function
Membrane Proteins
Most membrane functions are carried out by
membrane proteins – in animals, proteins constitute
50% of the mass of most plasma membranes
(remaining being lipid, relatively small amounts of
carbohydrates on glycolipids and glycosylated
proteins)
Examples of classes of proteins
Functional Class Protein Example Specific Function
Transporters Na+ pump Actively pumps Na+ out of cells and K+ in
Anchors Integrins Link intracellular actin filaments to
extracellular matrix proteins
Receptors Platelet-derived growth Binds extracellular PDGF and generates
factor (PDGF) receptor intracellular signals that cause the cell to
grow and divide
Enzymes Adenylyl cyclase Catalyses the production of intracellular
signalling molecule cyclic AMP in response
to extracellular signals
Membrane Proteins Associate with the Lipid Bilayer in Various ways – any or all may
contain sites of acylation or isoprenylation
1. Transmembrane proteins: extend through
bilayer, have hydrophobic regions lie in the
interior of the bilayer, nestled against the
hydrophobic tails of the lipid molecules –
hydrophilic regions exposed to the aqueous
environment on either side of the membrane:
can extend as a single α helix, multiple α helices or as a rolled up β sheet (β barrel)
- Vast majority of transmembrane domains are single, long alpha helices however
some bacterial proteins contain only beta strands
In the beta-strand extended conformation alternate amino acids have side chains
facing in opposite directions – for an aqueous solute channel polar residues will tend
to be on the inner faces and hydrophobic will be enriched on the outer faces
, In an alpha-helical structure amino acids align approximately every 3 or 4 residues
– a repeating pattern of hydrophobicity will occur amongst amino acids facing the
non-polar core of the bilayer
Specificity of protein function can then be encoded by supportingintra and
intermolecular peptide associations, specific interactions in the loops or in the
buried core of the protein itself
- To dissolve in the hydrocarbon, core of membranes these transmembrane spans
must be predominantly hydrophobic – to pass from one side to another, they must
be 27-30 angstoms in length (given a helical rise of 1.5 anstrongs per amino acid,
transmembrane helices are predominantly around 20 residue (typical6-7 amino
acids) stretches enriched in hydrophobic amino acids – thus very easy to spot in
genomic and other sequence databases: 20-25% genome codes transmembrane
Properties of membrane spanning amino acids (to make helices)
- Aliphatic side-chain: Isoleucine (I), Leucine (L), Valine(V)
- Aromatic side-chain: Phenylalanine (F), Tyrosine (Y), Tryptophan (W)
- Helical propensity: proline probably not common in helices (cannot form H-bonds
needed to form the shape)
- Some transmembrane proteins can have many transmembrane spans. Although few
have more than 14 (eg. adenylyl cyclase has 12) – domains are alpha helices which
“bundle up” to form a core (which can have functions – channels, receptors, anchors)
- Proteins contain loops which stick to
the aqueous phases surrounding
membranes
Hydropathy Plot
Assume we have a 500 long peptide –
Hydrohobicity
reading frame is 20 a.a, each time
shifting by one a.a, we can score the
hydrophobicity
Eventually peaks of hydrophobicity
will be observed
Used to resolve topology
Methods of ranking hydrophobicity affects plot, but still a good predictor of
topology
There needs to be a way to rank hydrophobicity – decide on hydropathy scales
, - Experimentally: elution from reverse phase HPLC columns, partition between water
and organic solvents
- Theoretically: sequence analysis of known transmembrane region
Topology – which orientations and why?
“Topology question” – applies to both
single and multi-pass transmembrane
proteins
“Positive inside rule” – most negatively
charged lipids are on the inside of the
plasma membrane, consequently, a cluster
of basic (positively charged) amino acids can
orientate the protein
Usually 1st and 2nd transmembrane spans are the most hydrophobic –
whichever way the first spans insert into the membranes may dictate orientation of
subsequent spans if “bundling up” occurs
Post-translational modification (including proteolysis) during the maturation of
protein can change apparent orientation
If Glycosylation sites are present, loops will eventually appear on the outside of the
cell (but on the inside of organelles where sugars are added)
To deduce correct topology…
Experimentally: raise antibodies to likely loop regions and stain intact and
permeablised cells, predict and then mutate possible glycosylation sites
Theoretically: study sequences of proteins of known function and topology that
have global and local similarities to your target
2. Other proteins located entirely in the cytosol, anchored to the inner leaflet of the
lipid bilayer by an amphipathic α helix exposed on the surface of the protein
3. Proteins lie entirely outside bilayer, on one side or the other, attached to the
membrane by one or more covalently attached lipid groups
Recruiting proteins to membranes (presence of PIPs recruits cytosolic proteins)
- Phospholipid phosphatidylinositol (PI) can be further phosphorylated 1-3 times to
produce a family of inter-convertible lipids = Polyphosphoinositides (eg. PI(4,5)P 2,
PI(3,4,5)P3)
- Some proteins containing discrete modules (pleckstrin homology domains – PH
domains) which can recognise and bind to specific PIPs
Membrane Proteins
Most membrane functions are carried out by
membrane proteins – in animals, proteins constitute
50% of the mass of most plasma membranes
(remaining being lipid, relatively small amounts of
carbohydrates on glycolipids and glycosylated
proteins)
Examples of classes of proteins
Functional Class Protein Example Specific Function
Transporters Na+ pump Actively pumps Na+ out of cells and K+ in
Anchors Integrins Link intracellular actin filaments to
extracellular matrix proteins
Receptors Platelet-derived growth Binds extracellular PDGF and generates
factor (PDGF) receptor intracellular signals that cause the cell to
grow and divide
Enzymes Adenylyl cyclase Catalyses the production of intracellular
signalling molecule cyclic AMP in response
to extracellular signals
Membrane Proteins Associate with the Lipid Bilayer in Various ways – any or all may
contain sites of acylation or isoprenylation
1. Transmembrane proteins: extend through
bilayer, have hydrophobic regions lie in the
interior of the bilayer, nestled against the
hydrophobic tails of the lipid molecules –
hydrophilic regions exposed to the aqueous
environment on either side of the membrane:
can extend as a single α helix, multiple α helices or as a rolled up β sheet (β barrel)
- Vast majority of transmembrane domains are single, long alpha helices however
some bacterial proteins contain only beta strands
In the beta-strand extended conformation alternate amino acids have side chains
facing in opposite directions – for an aqueous solute channel polar residues will tend
to be on the inner faces and hydrophobic will be enriched on the outer faces
, In an alpha-helical structure amino acids align approximately every 3 or 4 residues
– a repeating pattern of hydrophobicity will occur amongst amino acids facing the
non-polar core of the bilayer
Specificity of protein function can then be encoded by supportingintra and
intermolecular peptide associations, specific interactions in the loops or in the
buried core of the protein itself
- To dissolve in the hydrocarbon, core of membranes these transmembrane spans
must be predominantly hydrophobic – to pass from one side to another, they must
be 27-30 angstoms in length (given a helical rise of 1.5 anstrongs per amino acid,
transmembrane helices are predominantly around 20 residue (typical6-7 amino
acids) stretches enriched in hydrophobic amino acids – thus very easy to spot in
genomic and other sequence databases: 20-25% genome codes transmembrane
Properties of membrane spanning amino acids (to make helices)
- Aliphatic side-chain: Isoleucine (I), Leucine (L), Valine(V)
- Aromatic side-chain: Phenylalanine (F), Tyrosine (Y), Tryptophan (W)
- Helical propensity: proline probably not common in helices (cannot form H-bonds
needed to form the shape)
- Some transmembrane proteins can have many transmembrane spans. Although few
have more than 14 (eg. adenylyl cyclase has 12) – domains are alpha helices which
“bundle up” to form a core (which can have functions – channels, receptors, anchors)
- Proteins contain loops which stick to
the aqueous phases surrounding
membranes
Hydropathy Plot
Assume we have a 500 long peptide –
Hydrohobicity
reading frame is 20 a.a, each time
shifting by one a.a, we can score the
hydrophobicity
Eventually peaks of hydrophobicity
will be observed
Used to resolve topology
Methods of ranking hydrophobicity affects plot, but still a good predictor of
topology
There needs to be a way to rank hydrophobicity – decide on hydropathy scales
, - Experimentally: elution from reverse phase HPLC columns, partition between water
and organic solvents
- Theoretically: sequence analysis of known transmembrane region
Topology – which orientations and why?
“Topology question” – applies to both
single and multi-pass transmembrane
proteins
“Positive inside rule” – most negatively
charged lipids are on the inside of the
plasma membrane, consequently, a cluster
of basic (positively charged) amino acids can
orientate the protein
Usually 1st and 2nd transmembrane spans are the most hydrophobic –
whichever way the first spans insert into the membranes may dictate orientation of
subsequent spans if “bundling up” occurs
Post-translational modification (including proteolysis) during the maturation of
protein can change apparent orientation
If Glycosylation sites are present, loops will eventually appear on the outside of the
cell (but on the inside of organelles where sugars are added)
To deduce correct topology…
Experimentally: raise antibodies to likely loop regions and stain intact and
permeablised cells, predict and then mutate possible glycosylation sites
Theoretically: study sequences of proteins of known function and topology that
have global and local similarities to your target
2. Other proteins located entirely in the cytosol, anchored to the inner leaflet of the
lipid bilayer by an amphipathic α helix exposed on the surface of the protein
3. Proteins lie entirely outside bilayer, on one side or the other, attached to the
membrane by one or more covalently attached lipid groups
Recruiting proteins to membranes (presence of PIPs recruits cytosolic proteins)
- Phospholipid phosphatidylinositol (PI) can be further phosphorylated 1-3 times to
produce a family of inter-convertible lipids = Polyphosphoinositides (eg. PI(4,5)P 2,
PI(3,4,5)P3)
- Some proteins containing discrete modules (pleckstrin homology domains – PH
domains) which can recognise and bind to specific PIPs
