208B Membranes-Proteins

Question 1

How are membrane proteins classified?

Answer 1

based on how they associate w/the membrane

Question 2

What are the two main classes of membrane proteins?

Answer 2

1) The peripheral membrane proteins = found bound to the surface of the membrane but are not tightly associated w/the membrane
2) Integral membrane proteins = these are very TIGHTLY associated w/the membrane and the membrane must be disrupted to release the protein

Question 3

What protein subclasses fall under peripheral membrane proteins?

Answer 3

Within the class of peripheral membrane proteins are AMPHITROPIC proteins. These proteins are found free in the cytosol as well as bound to the membrane. Most often, their association is reversible and is regulated. E.g., phosphorylation or ligand‐binding can bring about a change in the conformation allowing the protein to bind to the membrane.

Question 4

How are peripheral membrane proteins bound to the surface of the membrane?

Answer 4

• lipid head-groups
• integral membrane proteins

Peripheral membrane proteins are bound to polar head groups or to other integral membrane proteins through electrostatic interactions or hydrogen bonds.

Question 5

How can peripheral membrane proteins be removed from the membrane?

Answer 5

They can be removed by mild treatment with the addition of urea or with changes in pH (such as by adding carbonate to increase the pH) and ionic strength. These proteins are quite stable and are soluble in the absence of lipids.

the membrane is left intact when these proteins are removed

Question 6

How are proteins anchored to the membrane?

Answer 6

through lipid linkages. 1) saturated acyl groups 2) isoprenoids 3) glycosyl phosphatidylinositol (GPI)

These covalent linkages are of several types including saturated long‐chain fatty acids, isoprenoids or oligosacchardide‐derivatives of phosphatidylinositol. The lipid linkages also serve a targeting function, directing a protein to its correct membrane location.

Question 7

What are the examples of saturated acyl groups (lipid linkage) provided in this lecture?

Answer 7

1) palmitic acid

2) myristic acid

Question 8

What is palmitic acid?

Answer 8

a 16‐carbon fatty acid that can be attached via a thioester linkage to a cysteine residue of the membrane protein. This is a reversible modification and is involved in intracellular signaling

Question 9

What is myristic acid?

Answer 9

a 14‐carbon fatty acid which is linked via an amide bond to the N‐terminal glycine. This is a stable attachment, and is not reversible

Question 10

What are the examples of isoprenoid groups provided in this lecture?

Answer 10

Thioester bond to C-terminal Cys (attach to C-terminal Cys residues via thioester bonds)

(farnesyl 15C and geranyl-geranyl 20C)

Question 11

What are glycosyl phosphatidylinositol or GPI anchors?

Answer 11

these anchors include inositol bound to a short oligosaccharide chain which covalently linked to a c-terminal residue through phosphoethanolamine.

Question 12

How does GPI differ from most peripheral membrane proteins?

Answer 12

Unlike most peripheral membrane proteins, GPI‐ anchored protiens are not removed by mild treatments. GPIs are usually found on the extracellular side of the plasma membrane. GPI‐anchored proteins can be released by phospholipase C.

Question 13

How can integral membrane proteins be extracted from the mebrane?

Answer 13

require a detergent

Question 14

How often do integral membranes span the membrane and how do they interact with the membrane?

Answer 14

Integral membrane proteins span the membrane either once as in glycophorin or multiple times (most commonly 7 or 12)

The region of the protein that is within the membrane is called the transmembrane region and is mostly comprised of hydrophobic residues. This hydrophobic region interacts with the lipid core.
These proteins also have a specific orientation in the membrane, giving a sidedness to the membrane. In the case of glycophorin, the glycosylated residues are found on the extracellular face of the bilayer.

Question 15

What type of conformation does the membrane spanning region of integral proteins adopt and why?

Answer 15

The membrane‐spanning regions typically adopt an α‐helical or β‐barrel structure, because these motifs maximize the hydrogen bonds within the hydrophobic membrane. I.e., since the membrane‐spanning regions cannot form H bonds with the solvent (e.g., water), they form H bonds within themselves.

Question 16

How many helices do integral membranes have?

Answer 16

Integral membrane proteins can have between 1 and 12 transmembrane helices (but usually 1, 7, or 12). The helices are comprised of ~ 20 residues which mainly have hydrophobic side chains.

Question 17

What can a hydropathy plot tell us about integral membranes?

Answer 17

A hydropathy plot shows the free energy of transfer of the specific amino acid from an organic solvent to water. By summing up the free energy of transfer for a set of residues within a sequence, one can calculate the hydropathy index for that sequence of amino acids. A high positive value for the free energy indicates that these residues do not favor transfer to water and hence would be hydrophobic. A stretch of hydrophobic residues indicates a putative transmembrane region. Hence these hydropathy plots can be uses to predict transmembrane regions within proteins.

Question 18

A high positive value for the free energy indicates what about the hydropathy of residues?

Answer 18

these residues do not favor transfer to water and hence would be hydrophobic

Question 19

How can a hydropathy plot tell us there is a transmembrane region?

Answer 19

A stretch of hydrophobic residues indicates a putative transmembrane region

Question 20

What is bacterial rhodopsin?

Answer 20

one of the better studied integral membrane proteins. It is comprised of 7 transmembrane regions which adopt an alpha helical structure. These helices are connected by non-helical loops at the inner and outer surfaces of the membrane.

Bacterial rhodopsin is a light-driven proton pump. Membrane

Question 21

For bacterial rhodopsin how can membrane-protein topology be determined?

Answer 21

using reagents that react w/the protein but do not cross the lipid bilayer. e.g., the protease trypsin cleaves the extracellular domains but does not affect the regions that w/in the bilayer or exposed to the inner surface.

Question 22

What is a helical bundle motif?

Answer 22

a type of motif that a certain proteins such as ion channels adopt.

Transmembrane helices come together for form a pore. A single helix may not have space w/in the helix for solutes to pass through but when several alpha helices come together they can enclose a pore that is large enough for solutes such as ions to pass through.

Question 23

Alpha helical bundle motifs (transmembrane pores) transport what?

Answer 23

used for the transport of water-soluble compounds.

Question 24

What are common structural motifs for integral transmembrane proteins?

Answer 24

1) Alpha helical bundle motifs

2) transmembrane beta barrels

Question 25

What characteristics do transmembrane beta barrels portray?

Answer 25

these barrel structures are made up of 20 or more transmembrane segments which are antiparallel.

Question 26

Why are transmembrane beta barrels stable structures?

Answer 26

b/c the maximal intrachain hydrogen bonding

Question 27

What can be transported through a beta barrel?

Answer 27

Beta barrels are polar on the inside and allow certain polar solutes to be transported, while the hydrophobic outside interacts with fatty acid components of the membrane.

Question 28

Where are transmembrane beta barrels observed?

Answer 28

in outer membrane of bacteria and in mitochondria and chloroplasts of eukaryotes

Question 29

How are alpha helix different than beta strands?

Answer 29

beta strands are more extended with 7 to 9 residues to span the membrane

Question 30

Which amino acids are present at the interface between lipid and water of integral membranes?

Answer 30

Trp and Tyr. These residues serve as membrane-interface anchors and interact both w/aqueous and the lipid phase.

Question 31

Which charged residues are found predominantly in the aqueous phase of transmembrane proteins?

Answer 31

Asp
Glu
Lys
Arg

Question 32

What is the positive inside rule?

Answer 32

a feature of transmembrane surfaces. Positively charged residues (Lys, Arg, His) occur more often on the cytoplasmic side of the membrane

Question 33

Question for you: Rationalize the presence Trp and Tyr at the lipid‐water interface, as well as the presence of the positively charged residues at the cytosolic surface.

Answer 33

idk - look at last slide of lecture