Membrane Proteins Flashcards
Proteins represent approximately how much of membrane mass?
50%
What is the purpose of membrane proteins?
to perform most of the membrane’s specific tasks
What are the 3 categories of membrane proteins? How are they organized?
integral membrane protein
peripheral membrane protein
lipid-anchored membrane protein
organized depending on their interaction with the membrane
All membrane proteins are hydrophobic, hydrophilic, or amphipathic?
amphipathic - contain both hydrophobic and hydrophilic regions
Describe integral membrane proteins
membrane proteins that penetrate the lipid bilayer mostly in the shape of alpha helices inside the lipid bilayer
Most of which cross the entire bilayer (transmembrane)
Describe transmembrane proteins
integral membrane proteins that cross the entire lipid bilayer
What is a single pass membrane vs. a multi-pass membrane?
single pass integral membranes cross the lipid bilayer once
multipass integral membranes cross the lipid bilayer more than once
Describe peripheral membrane proteins
Membrane proteins that either side/face of the membrane with non-covalent interactions (ie., they do not penetrate the lipid bilayer)
They are usually bound to an integral protein
Why can peripheral proteins be easily released from the membrane?
because they do not penetrate the lipid bilayer and can be removed by gentle extraction processes that disrupt the weak interactions (H bond or ionic) by changing the pH or salt concentration
Describe lipid-anchored proteins
membrane proteins that are located outside the lipid bilayer on either face and covalently linked to a lipid molecule within the bilayer
What is a common anchor for lipid-anchored proteins on the outer face of a membrane?
phosphatidylinositol linked to a set of sugars called GPI
What is GPI?
glycosylated PI
a small set of sugars that are linked to a phosphatidylinositol
What is a common anchor for lipid-anchored proteins on the inner face of a membrane?
a long hydrocarbon chain
Where does a transmembrane alpha helix protein have amino acids?
in 3 distinct regions:
- in the cytosol
- in the lipid bilayer
- in the extracellular space
What kind of amino acids does a transmembrane alpha helix protein have in each of its distinct regions
CYTOSOL:
polar amino acids
LIPID BILAYER:
non polar amino acids
ECM space:
polar amino acids
Approximately how many amino acids in a helix does it take to span the bilayer?
~20 hydrophobic amino acids
If a hydrophilic amino acid is in the core of the bilayer, its R group is usually somehow ____ or ___ with the charged ____ ____
Its R group is usually somehow SHIELDED from the hydrophobic conditions of the inside of the bilayer or INTERACTING with the charged PHOSPHOLIPID HEADS
T or F: of the 20 amino acids it takes to cross the lipid bilayer, most of them will be hydrophilic
FALSE. Most of them will be hydrophobic due to the hydrophobic conditions of the lipid bilayer
SOME may be hydrophilic but they would be shielded from the conditions or near the edges
What are 6 techniques for studying membrane proteins?
- Freeze fracture
- hydropathy plots
- cell fusion
- FRAP
- Single particle tracking
- detergent application
Describe the freeze fracture technique
An older technique of studying membrane proteins that allows scientists to understand the faces of the bilayer and the arrangement of the proteins
- tissue is frozen and cracked by hitting it with a knife
- The membrane will split at the weakest point = between the two monolayers and allow the scientist to pull the membrane faces apart
- Proteins will peel off with one face and leave a corresponding gap in the other face (because the membrane is frozen, the gaps will stay and will not be refilled with lipids)
- heavy metals are used to create a replica of the membrane faces so that the topography of the membrane can be studied under an electron microscope
What is the purpose of freeze fracture technique
Provides an understand of the two monolayers and how proteins are arranged within a membrane
Helped understand that membranes penetrate the lipid bilayer
What big model did the freeze fracture technique help establish?
the fluid mosaic model of membranes
Describe hydropathy plots
Each amino acid in an ALPHA HELIX protein is given a value based on its polarity ranging between -5 to 5.
Nonpolar/hydrophobic = positive polar/hydrophilic = negative
A long sequential series (~20) positive values may indicate a transmembrane alpha helix protein
Each long spanning peak is a transmembrane protein
What is the purpose of hydropathy plots?
Indicates which portions of transmembrane proteins are in the membrane
Helps us determine which proteins are membrane proteins and if there’s any transmembrane proteins
Useful in drawing membranes and proteins and understanding the overall structure of a membrane
Which kind of protein structure are hydropathy plots for?
Alpha helices NOT beta sheets
But most membrane proteins are alpha helices anyways
Describe cell fusion
A technique of studying membrane proteins that combines two different cell types to produce a single cell with a CONTINUOUS PLASMA MEMBRANE
proteins from each of the original cells are labeled and their movement is tracked over time
Over time, the new single cell will have proteins from both original cells that are UNIFORMLY DISTRIBUTED
What is the purpose of cell fusion?
Helps us understand how proteins move through the membrane by tracking their movement/diffusion throughout the new fused cell membrane
Describe FRAP (Explain what it stands for too)
Fluorescence Recovery After Photobleaching is a technique of studying membrane proteins that measures the lateral diffusion/movement rate of membrane proteins
- a specific protein of interest is tagged with something fluorescent:
- an antibody attached to a fluorophore
- a green fluorescent protein - a laser beam bleaches (kills) a small area of the membrane = no more fluorescence
- any recovery of fluorescence in the bleached area suggests LATERAL PROTEIN MOVEMENT and fluidity in the membrane
- the time it takes to fully recover is measured to get the rate of recovery
- faster recovery = more later protein movement
- no recovery = protein is fixed/no lateral movement
What is the purpose of FRAP?
to understand membrane fluidity and the lateral movement of proteins in the membrane
What does it say about the movement of proteins if the rate of recovery from FRAP is rapid?
There is lots of lateral protein movement
What does it say about the movement of proteins if the bleached area (FRAP) never recovers?
There is no lateral protein movement (ie., the protein is fixed)
T or F: FRAP is only for protein movement
False, it can be done to track lipid movement too
Describe single-particle tracking
A technique of studying membrane proteins that tags INDIVIDUAL membrane molecules with fluorescence molecules and tracks their movement with video
What is the purpose of single-particle tracking?
Gives us the movement of ONE membrane protein over time
It can tell us if there are limits to fluidity for proteins
T or F: single-particle tracking can only be used for protein movement
False, it can be done with membrane lipids too
What are some examples of reasons why a protein has limited lateral movement/diffusion in the membrane?
It is anchored permanently to the cytoskeleton = no movement
It is bound to the cytoskeleton = can only move along the cytoskeleton
It is stuck between other proteins
It is stuck in a cytoskeleton box = can only move within the small region of the box
It is stuck in the ECM
What are some limitations of lateral protein movement/diffusion in polar cells?
Due to the asymmetry of polar cells, the apical surface and basal surface are different
Some proteins may be restricted to the apical surface and some may be restricted to the basal surface
Movement may be blocked by intracellular junctions
ex. epithelial cells
Describe detergent application
A technique of studying membrane proteins that removes integral proteins from the lipid bilayer with an amphipathic detergent
VERY difficult to remove
What kind of detergent is required to remove what kind of proteins from the bilayer? Why does this work?
An amphipathic detergent is required to remove an integral protein from the bilayer
Amphipathic detergents can substitute the phospholipids and stabilize the integral protein while also making them soluble and therefore removable
What are the concerns with detergent application?
Removal of the protein from the bilayer may cause the protein structure to collapse because they require the membrane for their structure - may not refold again
Some detergents may disrupt the tertiary structure of proteins (ex. SDS)
