Jan 23- membrane proteins
What are the kinds of beta barrels?
- 8 stranded OmpA, 12-stranded OmpA, 16 stranded porin, 22-stranded FepA
What do amphipathic alpha-helix proteins associate with?
Both the hydrophobic lipid bilayer and the cytosol. One side is exposed to aqueous cytosol while the other side is lodged in the bilayer.
What is the RBC "ghost" experiment?
1. RBC is placed in hyptonic solution and water enters the cell until it expands and then bursts (shown visually as rbc inflated) 2. Then you can wash it (shown visually as a deflated rbc with a hole), wash and reseal (shown visually as a deflated RBC with no hole), or you can disrupt and reseal (shown as small circular vesicles which are closed and also can be inside out)
What are the kinds of transmembrane proteins?
1. Single alpha-helix membrane-spanning domain: hydrophobic alpha helix (e.g., glycophorin) 2. Multiple alpha-helix membrane-spanning domains: (e.g., 7-pass transmembrane proteins like bacteriorhodopsin) 3. Beta-barrel (e.g. porins)
How long is one turn of an alpha helix? And how is the helix shown visually?
3.6 amino acids, which is 0.54 nm Shown visually: oxygen in carboxyl group id hydrogen bonded to hydrogen in amino group, which will be 4 amino acids down the line. R chains are facing out of helix and exposed to the face of the hydrophobic chains of the membrane.
How do proteins move in SDS page?
A current is run through a gel and the proteins are separated by mass. The larger proteins move more slowly (band at the top), while larger proteins move more quickly (band at the bottom)
How long are alpha helices?
At least 20 amino acids long (typically 20-30)
How do detergents change nature in water at low concentration and higher concentrations? Why do they do this?
At low concentration, detergents in water exist as monomers and at higher concentrations (the critical micellar concentration) detergents form micelles (with the hydrophobic tails on the inside)
What are the common ways protein diffusion can be restricted?
Aggregation- shown visually as proteins clumped in separate regions of plasma membrane Binding to molecules in extracellular matrix-shown visually as clump of proteins bound to a band Binding to intracellular molecules- shown visually as clump of proteins bounded to band inside cell Binding to molecules on other cells- shown visually as proteins interfacing between cells
What is required for a protein to be able to transmit a signal from the extracellular space to the cytosol?
It has to span the entire membrane
Why can't a helix contain a proline?
It will break an alpha helix due to its rigid, cyclical side chain. Only 1 in the middle is allowed.
Which proteins and lipids are glycosylated?
Only those on the extracellular face of the plasma membrane
What is the glycocalyx?
The carbohydrate layer around the cell's perimeter which is formed from carbohydrates attached to transmembrane proteins and lipids
Where are carbohydrates found?
They are abundant on the plasma membrane; glycocalyx is formed of glycolipids, glycoproteins, and proteoglycans
What kind of molecules are transmembrane proteins?
They are amphipathic molecules
Do transmembrane alpha helices have to be hydrophobic for their initial insertion on the membrane?
Transmembrane alpha helices have to be hydrophobic for their initial insertion in the membrane, but their final conformation may depend on interactions between a few polar amino acids in the helices
What can the polar amino acids do?
They can create a channel for the passage of ions in the middle of the helix
Why are differentiated blood cells the perfect eukaryotic model for studying plasma membrane proteins?
They have no internal membrane-bound organelles
How do we study membrane proteins?
Using detergents: 1. Ionic detergents (e.g. SDS) 2. Non-ionic detergents (e.g. Triton X-100, B-octylglucoside)
Why do we take phospholipids out and add them back in for the solubilizing Na/K pump experiment?
Want pump to be in a vesicle so you have to add phospholipids
What is the acronym for determining if aa sequence is alpha helix?
FAMILY VW P and G are excluded.
How long are beta sheets?
Fewer than 10 amino acids
How are solubilizing experiments optimized?
For tiny liposomes with only 1 protein because you can't control whether the proteins face in or out. Use antibodies to direct protein direction.
How does the alpha helix sit in the phospholipid bilayer?
Hydrophobic amino acid side chains stick oit and make contact with hydrophobic core, but the hydrogen bonded back bone stays tucked inside
How could you determine an amphipathic alpha helix from an AA sequence?
If it alternates between hydrophobic and hydrophilic AA every 2-3 in the sequence
What is the experiment to show that proteins can diffuse with a membrane?
In a heterokaryon cell, mouse proteins green and human proteins red, let diffuse over plasma membrane and over time mouse cell proteins will aggregate togethet and human cell proteins aggreagate together
What are the characteristics of proteins anchored to the membrane by lipid linkages?
1. Can be attached to the cystolic face by fatty acid chains (myrisitic or palmitic acid) or prenyl groups (farnesyl or geranylgeranyl groups)
What are beta barrels?
Beta sheets which curve around to form cylinders; these barrels can also act as pores
How does the bacteriorhodopsin protein function?
-Photons of light cause a conformational change that pumps H+ from the inside to the outside of the cell -The photons hit a pigment called retinal -This sets up a proton gradient that drives production of ATP
What is the hydropathy index?
-The free energy change that occurs when a segment of a peptide chain is transferred to water, i.e. how hydrophobic the amino acids in that segment are
What do the peaks in a hydropathy index correspond to?
-The number of transmembrane domains, which means there is energy required to get it in an aqueous environment (unlike with hydrophilic molecules). In this way, a positive hydropathy index indicates hydrophobic amino acids. -peak must be wide -Glycophorin has 1 and bacteriorhodopsin has 7
What are the features of beta barrels?
-rigid; cant undergo conformational changes like alpha helices -generally restricted to the outer membranes of mitochondria, chloroplasts, and bacteria -center of barrel can be open- can allow passage of hydrophilic molecules
What is Fluorescence Recovery After Photobleaching (FRAP)?
1. Bleach proteins with laser beam 2. And then over time some of those proteins will recover and become unbleached
What are the features of detergents?
All are amphipathic molecules, they are cone-shaped, and form miscelles in water
How are amino acids charged in amphipathic alpha helixes?
Amino acids with hydrophobic side chains on one side and hydrophilic side chains on the other
How are proteins linked to the exoplasmic face of the membrane?
By glycophosphatidylinositol (GPI) anchors
How do detergents solubilize liquids and integral membrane proteins?
By interacting with hydrophobic and hydrophilic domains. The detergent miscelles surround the lipid or protein forming a complex, which are called mixed miscelles.
How can protein diffusion be restricted within the cell?
By junctions or other barriers. Protein A on one side of tight junction and protein B on other side of tight junction.
How is GPI anchor cleaved?
By phospholipase C
Which proteins are less soluble in SDS page?
Integral proteins are less soluble than peripheral proteins
Why is it important that membrane protein and lipid composition influence curvature of a membrane? How is it shown visually?
Intracellular organelles need to change shape to create vesicles that transport proteins and lipids to other parts of the cell. Shown visually: 1. Protein with hydrophobic domain inserted into only one leaflet of membrane. Looks like green clovers sticking out of one side. 2. Curved protein complexes bound to lipid head groups. Looks like a connected green range of peaks. 3. Lipids with larger head groups (e.g. PI) inserted on only one side of the membrane. Looks like a small connected green blob in the middle.
How do each of the anchors function?
Myristoyl anchor- amide linkage between terminal amino group (from protein) and myristic acid Palmitoyl anchor- thioester linkage between cysteine and palmitic group Farnesyl anchor- thioester linkage between cysteine (from C terminal end) and prenyl group
Are beta barrels hydrophobic? And how long are they?
No, R chains alternate between hydrophobic and hydrophilic. This would show up as zero on a hydropathy plot. About 10 AA
What is the polarity of almost all amino acids in an alpha helix?
Nonpolar
What are the functions of membrane carbohydrates?
Protection: protects cells from harsh extracellular conditions Cell-cell recognition events (e.g. white blood cells adhering to endothelial lining of blood vessels)
What are peripheral membrane proteins?
Proteins that are localized to the membrane by non-covalent interactions with integral membrane proteins; can be extracted by high salt or changes in pH
What is corticol cytoskeleton underling the RBC formed out of?
Spectrin and other proteins including glycophorin, band 3, and band 4.1