baker - protein structure

Question 1

which lipids are anchored at the n terminus and at the c terminus

Answer 1

acetylation at N-terminus

prenylation at C-terminus

Question 2

name the properties of acetylated proteins and give two names examples with their chain length

Answer 2

• Can link carboxylic acid of f/a to n-terminal glycine by amide bond
o C14 chain – myristylation
o C16 chain – acetylation of palmitic acid
• This is co-translational (after N-terminal Met removal)
• Irreversible
• Always on inner leaflet
• Designed to put protein near leaflet

Question 3

name the properties of prenylated proteins and give two names examples with their chain length

Answer 3

• Thioether link to C-terminal cysteine (CH2—S—CH2)
• Fatty acid is a polymer of isoprene molecules
o Farnesyl (C15 chain)
o Geranylgeranyl (C20 chain)
• Once f/a added to cys ten c-term a/a’s are removed post-transl
• GTPases use these to anchor to membrane
• Inner leaflet
• Irreversible
• Designed to put protein near leaflet

Question 4

name the properties of thioester linked proteins and give four names examples with their chain length

Answer 4

•	Thioesters much easier to cleave than ether
•	Cysteine joined to:
o	Palmitic acid (C16)
o	Myristate (C14)
o	Stearate (C18)
o	Oleate (C18 unsat)
•	Reversible by use of thioesterases – maybe useful for signalling
•	Post translational 
•	Inner leaflet

Question 5

name the properties of GPI anchored proteins

Answer 5

simplified: membrane lipid—carbohydrate—protein
Modify c-terminus with ethanolamine linked to oligosaccharide (mannose) linked to inositol of phosphatidyl inositol (standard lipid within leaflet)
Outer leaflet

Question 6

what is a lipobox? give its sequence

Answer 6

a cysteine residue beginning sequence which can be attached to a lipid
seq: K-A/S-G/A-C
(C is only a/a in lipobox, all others are cleaved from the N-term)

Question 7

define membrane topology in relation to organelles (single membrane and double membrane)

Answer 7

OUT - inside of organelle/outside of cell
IN - cytosol
double membrane compartments:
IN: matrix
OUT: IM space

Question 8

what is glycophorin a? which side is on the outside?

Answer 8

glycophorin a is a single pass alpha helical protein

N is on outside as it has glycosylation sites

Question 9

define a type I membrane protein

Answer 9

o N-terminal in exoplasmic space
o Signal sequence determines orientation
o Stops at stop-transfer anchor sequence (h.phobic α-helix residues)
o Helix residues transferred into membrane

Question 10

define a type II membrane protein

Answer 10

o C-terminal in exoplasmic space
o Translocon not open and +ve residues – locates NH3 in cytosol
o Signal-anchor sequence allows protein to be transferred into membrane

Question 11

define a type III membrane protein

Answer 11

o N-terminus in exoplasmic space
o Positive residues on C-terminus locates it in cytosolic space
o Alpha-helix allows protein to be located in membrane
no signal sequence

Question 12

define a type IV membrane protein

Answer 12

multipass protein

Question 13

name 3 methods of sequence analysis that may determine membrane protein topology

Answer 13

membrane spanning regions are alpha helices - kyte and doolittle plot
look for positive residues either side of the helix (K&R)
prediction of the N-terminal ss (h.phobic) - may show up as little peaks on K&D plot

Question 14

name 6 experimental methods of determining membrane protein topology

Answer 14

1) enzyme tags - reporter proteins
2) chemical modification
3) antibodies against protein epitopes
4) EM microscopy
5) N/C terminal tags with GFP
6) adding/removing helices (in multipass proteins) allows you to see which side the GFP is on
(proteases work on the cytosolic side)

Question 15

name 3 reporter proteins used in determining membrane protein topology

Answer 15

LacZ
Alkaline phosphatase
Beta-lactamase

Question 16

how is lacz used as a reporter protein in membrane protein topology experiments?

Answer 16

CYTOPLASMIC protein
only folded and active tetramer on inside
fuse lacZ to c-term - follow with X-gal - change to colourless when C-term is on outside

Question 17

how is alkaline phosphatase used as a reporter protein in membrane protein topology experiments?

Answer 17

PERIPLASMIC protein - N-terminal leader
only active in reductive environments - contains 2S-S
follow with X-gal colourless —> blue if PhoA is present

Question 18

how is beta-lactamase used as a reporter protein in membrane protein topology experiments?

Answer 18

beta-lactamase protects cells against beta-lactam antibiotics
periplasmic protein
fuse to C-terminus - follow activity on amp plates - wont grow when on cytosolic (IN) side

Question 19

what is N-glycosylation scanning?

Answer 19

euk proteins have Asn residues in (NxT/S) sequence glycosylated by oligosaccharide transferase
(in lumen)
delete potential glycosylation sites by site directed mutagenesis - assay for glycosylation by SDS/PAGE electrophoresis or mass spec
can also be used as a tag in c-terminal deletion studies (like GFP)

Question 20

what is cysteine mutagenesis scanning?

Answer 20

Use cys to attach reagents eg biotin/radiolabels
Key Cys residue left
All other native Cys mutated to Ser
Can use native cys/introduce cys - want to analyse 1 cys usually

Question 21

what do biotinmalemide, N-[14C]ethylmaleimide and fluoresceinmalemide have in common? name a chemical that doesnt have this property

Answer 21

they are all membrane permeable

unlike acetomaleimide disulphonate which is membrane impermeable

Question 22

how can a cys specific membrane permeable label and a cys specific membrane impermeable block determine membrane protein topology

Answer 22

add label to intact cell and purify - permeable tag visible
add block to protein then add label - label cannot stick as the cysteine is outside of the cell (and the label is on the inside)

Question 23

name 2 methods of chemical modification that can be used to determine membrane protein topology

Answer 23

cys mutagenesis scanning

N-glycosylation scanning