Structures of membrane proteins. Flashcards
What is the speed of ATP powered pumps?
10^0 to 10^3 ions per second.
What is the speed of ion channels?
10^7 to 10^8 ions per second.
What is the main feature of a uniporter?
Moves substance with concentration gradient.
What is the main feature of a symporter?
Moves 1 substance against the concentration gradient and 1 with it. Move in the same direction
What is the main feature of a antiporter?
Moves 1 substance against the concentration gradient and 1 with it. Move in opposite directions.
What proved that aquaporins moved water into the cell?
When a frog lays a oocyte in freshwater the cell does not burst. When mRNA for aquaporins are placed into the cell the cell bursts.
What is in each subunit of an aquaporin?
6 transmembrane helices and 2 short helices per subunit.
What is the order of short and long subunits in the aquaporin?
Long, long, short, long.
How many subunits make up a aquaporin?
8.
How many pores are found in an aquaporin?
- Each subunit has a pore.
How is it possible for the short helices in the aquaporin to span the membrane?
Each one of the short helices goes into one of the membranes and together they can span the membrane.
What parts of the aquaporin are hydrophilic?
The top and the bottom.
How large is the aquaporin pore?
2.8A.
What is the aquaporin too small for?
H3O+.
What two highly conserved residues form the gate of the aquaporin?
Arg195 and His180. Both are these are positive so repel H3O+ residues.
What makes the transport of water through the aquaporin very fast?
The pore is lined with hydrophobic residues.
Explain why no protons are co translated in the aquaporin.
The proton wire is distributed by two Asn residues acting as proton donors.
When a proton wire is in place in the aquaporin what are all water molecules acting as?
Proton donors and acceptors.
What two processes allowed the determination of the Asn residues in the aquaporin?
X ray crystallography and electron microscopy.
What is the electrochemical potential gradient made up of?
Concentration and electron gradient.
What sets up the electron gradient?
K+ channel.
What sets up the chemical gradient?
K+/Na+ pump.
What does the Na+/lysine symporter do?
Pumps lysine up hill using the Na+ gradient.
What three things make up the single subunits in the potassium ion channels?
5s helix outside, 56 helix inside, P loop.
What two things make up the P loop in the single subunits in the potassium ion channel?
P segment and the selectivity filter.
What part of the of the potassium ion channel subunit is partially embedded in the membrane?
The P loop.
How many subunits make up the potassium ion channel?
4.
There are four subunits in the potassium ion channel. How many pores does this result in?
1.
What part of the K channel is homologous in all K channels?
P segment.
What is the role of the p segment in the K channel?
Imparts selectivity.
What does a mutation in the P loop of the K+ channel result in?
Loss of selectivity.
If the P segment in the bacterial P loop is mutated can not be replaced by a mammalian P segment. True or false?
False.
What is the transport rate of the K+ channel?
10^8 ions per second.
What is the empty space in the middle of the K+ channel called?
Vestibule.
What is the diameter of the vestibule in the K+ channel?
10A.
What is the diameter of the P loop/selectivity channel in the K+ channel?
3A.
What part of the K+ channel is hydrophobic?
Selectivity channel.
What part of the K+ channel is hydrophilic?
Vestibule.
Na+ is smaller than K+ but the K+ channel can still distinguish between the two. How?
Both ions are found in the solvated forms. Ka+ coordinates to 8 water molecules and Na+ coordinates to 6. Both of these need to be stripped. This normally costs energy but due to the arrangement of the carbonyl main chains in the selectivity factor this energy can be regained by the formation of 8 bonds, meaning it is only energetically favourable for Ka+ to enter the channel.
Is the desolvation of Na+ or K+ greater?
Na+.
What is the desolvation energy of Na+?
-301.
What is the desolvation energy of K+?
-230.
How many binding sites are there for K+ in the K+ channel?
4.
Energy increases in the binding sites in the K+ channel as you go down the channel. True or false?
False, they is only a small energy difference between the sites.
When travelling through the K+ channel/ selectivity filter K+ions fill the binding sites in turn. True or false?
False. They alternate between 1 and 3 and 2 and 4.
What drives K+ through the K+ channel/ selectivity filter?
Electrostatic repulsion of ions.
Movement through the K+ channel/ selectivity filter can been two states (1 and 2) What are these?
State 1: 1 ,3
State 2: 2, 4
What face are the N and C terminals on in the K+ ion channel?
Cytosol.
What joins the two transmembrane helices in the K+ channel?
The P loop.
What minimises the distance that the K+ has to move through the hydrophobic membrane?
The large vestibule.
The inside of compartmentalised organelles is classed as the outside. True or false?
True.
What are the key features of a type 1 membrane protein?
COO- in and NH3+ out. NH3+ contains a signal sequence.
What are the key features of a type 2 membrane protein?
NH3+ in and COO- out.
What are the key features of a type 3 membrane protein?
COO- in and NH3+ out. NO signal sequence.
What are the key features of a type 4 membrane protein?
Multipath
What are the key features of a GPI membrane protein?
NH3+ out, no cytosol domain.
What can the stop anchor sequence be describe as?
Hydrophobic.
What terminus of a protein is made first?
N terminus.
What happens when the stop anchor sequence is reached?
The translocation channel opens and allows it to enter the membrane.
What is an example of a type 1 membrane protein?
LDL receptors.
What is an example of a type 2 membrane protein?
Transferrin receptors.
What is an example of a type 3 membrane protein?
Cytochrome P450.
What is an example of a type 4 membrane protein?
G-protein coupled receptors.
What is an example of a type 5 membrane protein?
Plasminogen activation receptors.
What two amino acid residues are found in a type two signal pass outside the membrane?
Positive/ basic lysine and arginine residues.
What three things can you do during analysis the sequence of the membrane protein?
- Try to predict the membrane spanning helices from the hydropathy plot.
- Look for positive residues either side of the membrane ‘ positive inside rule’.
- Predict N terminal signal sequence.
What residues is the signal sequence made from?
Run of hydrophobic residues flanked by positive residue.s
The signal sequence is not long enough for it to be a transmembrane helix. How long is it?
10-18 residues.
What is the difference between a STA and a SA?
A STA is a internal stop-transfer anchor sequence and aa SA is a internal signal anchor sequence.
What key points did sequence analysis show about type one proteins? (3)
- Signal sequence.
- One STA.
- NH3+ lumen.
What key points did sequence analysis show about type two proteins? (3)
- Positive charge in cytosol next to the SA.
- One SA.
- NH3+ cytosol.
What key points did sequence analysis show about type three proteins? (3)
- Postive charge in cytosol next to the SA.
- One SA.
- NH3+ lumen.
What key points did sequence analysis show about type four A proteins? (3)
- SA/ STA/ SA/ STA
- Postive charge in cytosol next to SA.
- NH3+ cytosol.
What key points did sequence analysis show about type four B proteins?
- SA/ SA/ STA/ SA/ STA/ SA/ STA
- Postive charge in cytosol next to SA.
- NH3+ lumen.
What experimental tags can be used to determine the positioning of the membrane protein?
- Enzyme tags- reporter proteins.
- Chemical modification.
- Antibodies against protein epitopes.
- Microscopy (EM).
- N or C terminal tags with GFP.
- Protease accessibility.
You can use C terminal deletions to study membrane proteins but not N. Why?
The signal sequence is found at the N terminus.
How many subunits is reporter protein LacZ made from?
4.
How can LacZ be used as a reporter protein with membrane proteins?
LacZ is active in the cytoplasm as it is folder correctly. Can add it to the C terminus and follow the activity using Xgal sugar (blue when active.)
What type of protein is the reporter protein is PhoA alkaline phosphatase?
Periplasmic.
Where is reporter protein PhoA alkaline phosphatase expressed?
In prokaryotes, however it can be used as a reporter in prokaryotes and eukaryotes.
How many subunits make up the reporter protein PhoA alkaline phosphatase dimer?
2.
Why can the reporter protein PhoA alkaline phosphatase carry out its function?
It contains disulphides which are formed in the oxidative conditions of the periplasm but are not formed in the cytoplasm- making the reporter teportein inactive. tThis results in no phosphate bring produced and X-P not being activated (blue when active). The reporter protein is fussed to the C terminus.
What type of protein is the reporter protein B-lactamase?
Periplasmic.
What is the structure of the reporter protein B-lactamase?
Monomeric.
What is the role of B-lacatamase?
It prevents cells against the action of B lactic antibodies such as ampicillin.
How can B-lactamase be used as a reporter protein?
It can be fused to the C terminal of the protein and activity can be followed with the growth of cells on amp plates.
Eukaryote proteins are N-glycoslated in the ER. What does this?
Ogliosaccharide transferases (OST).
What does N glycosaltion involve?
Sugar groups added to ASN in NxT/S sequence in the luminal loop of the membrane protein.
How can potential glycosalation sites be detected?
SDM assay for glycosalation site by SDS/phage electrophoresis or mass spec to detect site sites (glycosaltion sites 2-5kd bigger.)
What can N-glycosaltion scanning also be used for in addition to adding sugars to ASN in the NxT/S sequence?
C terminal deletion of fusion proteins. Can link the glycosaltion domain to protein being studied.
Where must the glcyoslation site be for OST to work?
12 residues upstream or 14 residues downstream of the membrane.
What does cysteine mutagenesis scanning involve?
The addition of a reactive thiol group to cys mutants of the membrane proteins. Membrane then probed with membrane impermeable and membrane impermeable blocks. The mutated cys must first be changed into a serine.
Is the mutant protein in cysteine mutagenesis scanning usually active?
Yes.
What reagents can be used in cystitis mutagenesis scanning?
Biotin, fluorescent molecules or radiolabels.
What are three examples of a membrane permeable probe used in cysteine mutagenesis scanning?
- Biofinmalemide.
- N-[C14]ethlymaleimide.
- Fluroesceinmalemide.
What is an example of a membrane impermeable probe used in cysteine mutagenesis scanning?
Acetomalemide disuphonate.
What are thee advantages of cysteine mutagenesis scanning?
- Protein still active.
- In vivo studies possible.
- Small changes made to the protein.
What 4 methods can be used to determine membrane topology other than the scanning methods?
- Antibodies raised to natural or engineered epitopes.
- Immune affinity chromatography.
- GFP/ fluorescence microscopy.
- EM to directly visualise protein.
How can EM be used to determine membrane topology?
Protein labelled with gold antibody and particles coasted with protein A- this non specifically binds to the FC region.
