baker - protein structure Flashcards
which lipids are anchored at the n terminus and at the c terminus
acetylation at N-terminus
prenylation at C-terminus
name the properties of acetylated proteins and give two names examples with their chain length
• 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
name the properties of prenylated proteins and give two names examples with their chain length
• 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
name the properties of thioester linked proteins and give four names examples with their chain length
• 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
name the properties of GPI anchored proteins
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
what is a lipobox? give its sequence
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)
define membrane topology in relation to organelles (single membrane and double membrane)
OUT - inside of organelle/outside of cell IN - cytosol double membrane compartments: IN: matrix OUT: IM space
what is glycophorin a? which side is on the outside?
glycophorin a is a single pass alpha helical protein
N is on outside as it has glycosylation sites
define a type I membrane protein
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
define a type II membrane protein
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
define a type III membrane protein
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
define a type IV membrane protein
multipass protein
name 3 methods of sequence analysis that may determine membrane protein topology
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
name 6 experimental methods of determining membrane protein topology
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)
name 3 reporter proteins used in determining membrane protein topology
LacZ
Alkaline phosphatase
Beta-lactamase
how is lacz used as a reporter protein in membrane protein topology experiments?
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
how is alkaline phosphatase used as a reporter protein in membrane protein topology experiments?
PERIPLASMIC protein - N-terminal leader
only active in reductive environments - contains 2S-S
follow with X-gal colourless —> blue if PhoA is present
how is beta-lactamase used as a reporter protein in membrane protein topology experiments?
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
what is N-glycosylation scanning?
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)
what is cysteine mutagenesis scanning?
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
what do biotinmalemide, N-[14C]ethylmaleimide and fluoresceinmalemide have in common? name a chemical that doesnt have this property
they are all membrane permeable
unlike acetomaleimide disulphonate which is membrane impermeable
how can a cys specific membrane permeable label and a cys specific membrane impermeable block determine membrane protein topology
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)
name 2 methods of chemical modification that can be used to determine membrane protein topology
cys mutagenesis scanning
N-glycosylation scanning