Membrane Proteins

Question 1

Proteins represent approximately how much of membrane mass?

Answer 1

50%

Question 2

What is the purpose of membrane proteins?

Answer 2

to perform most of the membrane’s specific tasks

Question 3

What are the 3 categories of membrane proteins? How are they organized?

Answer 3

integral membrane protein

peripheral membrane protein

lipid-anchored membrane protein

organized depending on their interaction with the membrane

Question 4

All membrane proteins are hydrophobic, hydrophilic, or amphipathic?

Answer 4

amphipathic - contain both hydrophobic and hydrophilic regions

Question 5

Describe integral membrane proteins

Answer 5

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)

Question 6

Describe transmembrane proteins

Answer 6

integral membrane proteins that cross the entire lipid bilayer

Question 7

What is a single pass membrane vs. a multi-pass membrane?

Answer 7

single pass integral membranes cross the lipid bilayer once

multipass integral membranes cross the lipid bilayer more than once

Question 8

Describe peripheral membrane proteins

Answer 8

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

Question 9

Why can peripheral proteins be easily released from the membrane?

Answer 9

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

Question 10

Describe lipid-anchored proteins

Answer 10

membrane proteins that are located outside the lipid bilayer on either face and covalently linked to a lipid molecule within the bilayer

Question 11

What is a common anchor for lipid-anchored proteins on the outer face of a membrane?

Answer 11

phosphatidylinositol linked to a set of sugars called GPI

Question 12

What is GPI?

Answer 12

glycosylated PI

a small set of sugars that are linked to a phosphatidylinositol

Question 13

What is a common anchor for lipid-anchored proteins on the inner face of a membrane?

Answer 13

a long hydrocarbon chain

Question 14

Where does a transmembrane alpha helix protein have amino acids?

Answer 14

in 3 distinct regions:

1. in the cytosol
2. in the lipid bilayer
3. in the extracellular space

Question 15

What kind of amino acids does a transmembrane alpha helix protein have in each of its distinct regions

Answer 15

CYTOSOL:
polar amino acids

LIPID BILAYER:
non polar amino acids

ECM space:
polar amino acids

Question 16

Approximately how many amino acids in a helix does it take to span the bilayer?

Answer 16

~20 hydrophobic amino acids

Question 17

If a hydrophilic amino acid is in the core of the bilayer, its R group is usually somehow ____ or ___ with the charged ____ ____

Answer 17

Its R group is usually somehow SHIELDED from the hydrophobic conditions of the inside of the bilayer or INTERACTING with the charged PHOSPHOLIPID HEADS

Question 18

T or F: of the 20 amino acids it takes to cross the lipid bilayer, most of them will be hydrophilic

Answer 18

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

Question 19

What are 6 techniques for studying membrane proteins?

Answer 19

1. Freeze fracture
2. hydropathy plots
3. cell fusion
4. FRAP
5. Single particle tracking
6. detergent application

Question 20

Describe the freeze fracture technique

Answer 20

An older technique of studying membrane proteins that allows scientists to understand the faces of the bilayer and the arrangement of the proteins

1. tissue is frozen and cracked by hitting it with a knife
2. The membrane will split at the weakest point = between the two monolayers and allow the scientist to pull the membrane faces apart
3. 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)
4. 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

Question 21

What is the purpose of freeze fracture technique

Answer 21

Provides an understand of the two monolayers and how proteins are arranged within a membrane

Helped understand that membranes penetrate the lipid bilayer

Question 22

What big model did the freeze fracture technique help establish?

Answer 22

the fluid mosaic model of membranes

Question 23

Describe hydropathy plots

Answer 23

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

Question 24

What is the purpose of hydropathy plots?

Answer 24

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

Question 25

Which kind of protein structure are hydropathy plots for?

Answer 25

Alpha helices NOT beta sheets But most membrane proteins are alpha helices anyways

Question 26

Describe cell fusion

Answer 26

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

Question 27

What is the purpose of cell fusion?

Answer 27

Helps us understand how proteins move through the membrane by tracking their movement/diffusion throughout the new fused cell membrane

Question 28

Describe FRAP (Explain what it stands for too)

Answer 28

Fluorescence Recovery After Photobleaching is a technique of studying membrane proteins that measures the lateral diffusion/movement rate of membrane proteins 1. a specific protein of interest is tagged with something fluorescent: - an antibody attached to a fluorophore - a green fluorescent protein 2. a laser beam bleaches (kills) a small area of the membrane = no more fluorescence 3. any recovery of fluorescence in the bleached area suggests LATERAL PROTEIN MOVEMENT and fluidity in the membrane 4. 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

Question 29

What is the purpose of FRAP?

Answer 29

to understand membrane fluidity and the lateral movement of proteins in the membrane

Question 30

What does it say about the movement of proteins if the rate of recovery from FRAP is rapid?

Answer 30

There is lots of lateral protein movement

Question 31

What does it say about the movement of proteins if the bleached area (FRAP) never recovers?

Answer 31

There is no lateral protein movement (ie., the protein is fixed)

Question 32

T or F: FRAP is only for protein movement

Answer 32

False, it can be done to track lipid movement too

Question 33

Describe single-particle tracking

Answer 33

A technique of studying membrane proteins that tags INDIVIDUAL membrane molecules with fluorescence molecules and tracks their movement with video

Question 34

What is the purpose of single-particle tracking?

Answer 34

Gives us the movement of ONE membrane protein over time It can tell us if there are limits to fluidity for proteins

Question 35

T or F: single-particle tracking can only be used for protein movement

Answer 35

False, it can be done with membrane lipids too

Question 36

What are some examples of reasons why a protein has limited lateral movement/diffusion in the membrane?

Answer 36

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

Question 37

What are some limitations of lateral protein movement/diffusion in polar cells?

Answer 37

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

Question 38

Describe detergent application

Answer 38

A technique of studying membrane proteins that removes integral proteins from the lipid bilayer with an amphipathic detergent VERY difficult to remove

Question 39

What kind of detergent is required to remove what kind of proteins from the bilayer? Why does this work?

Answer 39

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

Question 40

What are the concerns with detergent application?

Answer 40

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)