Analysis of membrane components - lect 3-5 Flashcards
1
Q
what is the downside of having to use detergents to study integral membrane proteins?
A
detergent destabilizes proteins and can change their structure
2
Q
what are the 2 techniques used to find out analyse the TOPOLOGY of membrane proteins?
A
- glycosylation mapping
- natural/artificial epitope-tag
3
Q
what can mapping active glycosylation help us find out?
A
the glycosylated sites of a protein, aka the extracellular site (because glycosylation is always in extracellular side)
4
Q
what residue (aa) get glycosylated?
A
N (Asparagine), R (arginine), S (serine), T (threonine), Y (tyrosine)
5
Q
what is the procedure for glycosylation mapping?
A
- mutagenize (KO) putative (tentative) glycosylation sites
- determine which site is glycosylated by comparing the mutants vs WT
6
Q
what were the results of comparing mutated adenylate cyclase 9 at N residues to WT adenylate cyclase 9?
A
the mutants ran FASTER in the gel than WT because their glycosylated sites were mutated, therefore they were not glycosylated
7
Q
what do epitope tags allow us to discover?
A
detecting signal by fluorescent microscopy; it can allow us to know if the epitope is intracellular of extracellular
8
Q
what is the procedure for using epitope tags?
A
- mutagenesis: insert an epitope in your protein which you have an antibody for
- express the protein in a cell line
- expose the cells to the antibody
- detect the signal by fluorescent microscopy or by FACS
9
Q
give examples of epitopes that can be used
A
- natural
- HA tag
- Myc tag
- Flag tag
- V5 tag
- 6X His tag
- GST tag
10
Q
what happens if the epitope is located intracellularly?
A
2 options:
- no signal because the Ab can not enter the cell
- signal detection if the membrane is slightly permeabilized with detergent
11
Q
name the methods used for protein localization
A
- FRAP
- Immunofluorescence and FACS
- GFP-localization
- Cell surface biotinylation
12
Q
what is FRAP?
A
fluorescence recovery after photobleaching: method to study membrane dynamics in live cells
13
Q
what is the procedure for FRAP?
A
- shine a high energy laser beam (bleach) on a well-defined spot in fluorescently-labelled cells
- Allow the fluorescence to recover and monitor the recovery time
- repeat in different conditions
14
Q
what kind of proteins take more time to recover from bleaching?
A
proteins take longer than lipids
15
Q
why do proteins recover from bleaching slower than lipids?
A
proteins are less mobile;
they can be associated with the cytoskeleton and involved in focal adhesion
16
Q
what % of lipids are mobile?
A
100%, and they diffuse fast
17
Q
what is the definition of immunofluorescence?
A
IF: Immuno-affinity method that allows to study component cellular localization by microscopy
18
Q
what is FACS?
A
flow cytometry = fluorescence-activated cell sorting of immuno-labelled material
19
Q
what are the first steps of immunofluorescence AND FACS?
A
- express your protein in a cell line (dead)
- incubate your cells with a fluorescently-tagged antibody for your protein
- counterstain other cell compartments with another fluorescent dye
20
Q
what differentiates IF from FACS?
A
IF = you then just examine your fluorescent cells via IF or confocal microscope
FACS = you then quantify the immunofluorescence using FLOW CYTOMETRY
21
Q
how does flow cytometry work?
A
pass your cell sample in a cell sorter that has a detector that measures the fluorescence of each cell
22
Q
what does FACS (flow cytometry) allows you to quantify?
A
- the % of cells expressing your protein of interest
- the % of antigen per cell
23
Q
compared to IF, FACS does not give you information about what?
A
about cellular localization
24
Q
you get a graph when measuring FACS. how do you interpret the graph?
A
more scatter / granulation on the graph = more complex cell
25
what is GFP?
Green fluorescent protein (GFP) = protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue range
26
how can you tag multiple proteins at a time?
by using analogues of GFP such as YFP and CFP that emit fluorescence at other wavelengths
27
what do you need to be careful for when using GFP?
that the fusion did not alter the protein's function and/or cellular localization
28
what is cell-surface biotinylation?
Addition of a modified biotin molecule to cell surface proteins
29
what are the principles on which cell-surface biotinylation rely?
- Sulfo-NHS-Biotin derivative does not cross the plasma membrane
- Chemically modified biotin make it reactive to covalently bind to primary amines (NH2)
30
what is steptavidin? what is it used for?
a protein with very high affinity to biotin: can be used to pull down or analyze biotinylated proteins
31
what is required at cell surface for chemical reaction to primary amines (NH2 / mainly lysines) ?
free lysines
32
at what temperature does internalization stop?
4 deg celsius
33
sulfo-NHS-biotin and the protein of interest become ______ bound via primary _______
covalently;
amines (NH2)
34
what do you do after biotinylating your protein?
solubilise and precipitate the proteins
35
name different processes you can study via biotinylation
surface biotinylation, endocytosis biotinylation, re-insertion biotylation
(basically LOCALIZING the protein)
36
what is protein complementation assay?
Method of identification of protein-protein interactions based on the association of two proteins that generate a detectable readout
37
what are "bait" and "prey" in the protein complementation assay?
bait: part of the reporter protein that bind the protein of interest
pray: part of reporter protein that binds to a library of unknown proteins to see which ones interact with the protein of interest
38
name reporter proteins
- DHFR
- Beta-lactamase
- eGFP
- Horseradish peroxidase
- Luciferase
39
how does "survival selection" in the protein complementation assay work?
the bacteria that survive are those in for which the complementation of bait and prey protein worked (allows you to find what protein interacts with yours of interest)
40
explain the Protein Complementation Assays: Yeast 2 Hybrid system
GAL4 is a TF:
- bait protein expressed as a fusion to the GAL4 DNA binding domain
- prey expressed as fusion to GAL4 activating domain
- only when bait and prey interact can GAL4 function and bind to reporter genes which allows growth on a medium LACKING HISTIDINE
41
how do you confirm that the protein you found that interact with your protein of interest are not just artefacts?
1. classify the preys based on their function
2. verify by functional assay
42
what is co-ip?
Co-precipitation of protein(s) that interact with your protein of interest
43
what are the steps of co-ip?
1. solubilize your proteins with non-denaturing detergent
2. add anti-A ab and then ab-binding bends, and precipitate the complex by centrifugation
3. release the protein complex from the beads and run on SDS-page and western blot OR MS
44
what are limitations of co-ip?
- protein interactions must resist detergent solubilization
- hard from proteins embedded in rafts
- repeat procedure with irrelevant Abs to confirm
- don't know if interaction is direct
45
what are the requirements for an interaction to be able to resist detergent solubilization?
- interaction must occur outside a hydrophobic region
- interaction must occur through polar forces
46
what is a variant of co-ip for when there is no specific ab for out WT protein of interest?
using a mutant epitope-tagged protein of interest so it can bind our Ab
47
what is cross-linking?
chemical linking reaction between two nearby proteins using a bis-functional chemical reagent
48
describe a bis-functional reagent
2 reactive groups linked by a spacer arm
49
what is cross-linking good to study?
to study weak or transient protein-protein interactions, because cross-linking stabilizes them
50
the length of the spacer arm determines what?
the radius of action of th cross-linking agent
51
how does the cross-linking reaction happens?
via the primary amines (lysine side chain) of the proteins of interest
52
how can you study your cross-link once the reaction is done?
you can solubilize the proteins, immunoprecipitate, and analyze by western blot or mass spec.
53
what characteristics can cross-linkers have?
can be impermeant (charged) or permeant (no charge);
can be cleavable or (s-s bond) or not cleavable
54
what is the Bio-ID method?
Protein-protein interaction detection method using the fusion of a protein of interest with the biotin ligase BirA
55
how does bio-ID work?
fuse your protein of interest with BirA in a vector;
transfect the plasmid in cells;
add biotin (will bind to BirA);
lyse cells;
use streptavidin-coated beads to pull down biotinylated proteins only and identify them by mass spec.
56
what is tricky about Bio-ID?
it only labels proteins in the vicinity of your protein of interest and doesn't guaranty that the proteins make direct contact
57
what is FRET and BRET?
fluorescence / bioluminescence Resonance Energy Transfer
58
what does FRET/BRET allow us to do?
detect the proximity between fluorescent molecules based on their ability to transfer energy from a donor to an acceptor - confirm protein interaction that is already known
59
in what case with you see luminescence in FRET/BRET?
if proteins A and B interact, CFP will transfer energy to YFP (YFP absorbs the resonance energy from CFP and then releases it)
60
what are the conditions for FRET/BRET to function?
- The acceptor’s absorption spectrum must approximately match the donor’s fluorescence spectrum
- the two molecules must be in very close proximity
61
The efficiency of energy transfer in FRET is inversely proportional to what?
the 6th power of the distance
62
what is the formula for FRET efficiency?
E = 1 / (1 + R/R0)^6 )
63
what is R0? (in FRET)
Forster length = the Donor-Acceptor distance at which the efficiency of energy
transfer is 50% (greater overlap of absorption vs fluorescence spectrum = greater R0)
64
typically, the energy transfer of FRET works well in what radius?
R0/2 to 2R0 (usually 1-12 nm)
65
what constants / values of protein interaction can you determine using FRET? how?
via ligand association assay:
- affinity constant (Ka)
- maximal binding (Bmax)
via ligand competition assay:
- dissociation constant (Kd)
66
what is a ligand competition assay with FRET?
if there is competition between ligands, the FRET signal will decrease as we increase the competitor ligand concentration
67
difference between FRET and BRET?
for bioluminescence, you dont need to shine light because the first molecule (Coelenterazine) is luminescent
68
what is the crude method for LIPID analysis?
total extraction
69
what does an organic solvent do?
separates the hydrophobic molecules (lipids) from the aqueous phase
70
what is a TLC plate?
thin layer chromatography
71
how does TLC work?
separate molecules based on their affinity for a solvent (aka how polar they are)
72
how does liquid chromatography work?
measures the affinity of a protein / lipid to the liquid phase compared to the solid phase
72
name the steps of total lipid extraction
1. whole cell
2. treat with organic solvent (lipids go i organic phase)
3. migrate on a TLC plate
4. lipidomics
5. quantify, analyze data
73
what are the advantages and disadvantages of total lipid extraction?
Advantage: Cheap, rapid
Disadvantage:
- No information on organelles
- No information on asymmetry
74
what do phospholipases do?
they cleave phospholipids on the outer leaflet of the bilayer (chops the phospho head group)
75
how can you use phospholipases to study lipids composition?
calculate the ratio of lipids without phospho head group after incubating cells with phospholipases will tell you the % of said lipid that s in the outer vs inner leaflet
76
what is FITC-Annexin V used for?
when tagged with a fluorescent protein, it can be used as a marker for phagocytosis because it binds PS and high PS in the outer leaflet = apoptotic cell
77
what are the steps of immunoprecipitation
1. cell lysate using a non-denaturing detergent
2. add antibody: antigen-antibody complex
3. add beads: antigen-antibody-bend complex
4. wash and elute
5. purify the complex
6. use a denaturing detergent to study the individual proteins
78
what kind of detergent is SDS?
a denaturing detergent
79
what method can you use to study a whole organelle?
sub-cellular fractionation
80
why is 100% pure isolated membranes practically impossible to achieve?
because membrane disruption breaks up organelles, scrambles the 2 leaflets, causes fusion or adhesion of different membranes
81
what do non-denaturing detergents do to cell membranes?
disrupts the membranes and takes the phospholipids away
82
what can EDTA do?
prevents membranes from associating together (controls Ca2+ concentration)
83
why are proteases inhibitors important when doing membrane isolation?
because proteins could get degraded by the lysosomal proteases released when lysosome gets broken in the centrifuge
84
In absence of lipids, what happens to an amphiphilic peptide?
they aggregate together via hydrophobic patches when in a hydrophilic environment
85
what's the definition of a detergent?
Amphiphilic molecule which, when dissolved in water, is used to solubilize other molecules
86
what is cmc?
critical micelle concentration: the saturating concentration of detergent monomer = the concentration of detergent at which micelles first appear
87
why do detergents form micelles while phospholipids form bilayers?
because of their shape (triangular shape of detergent amphipathic molecules)
88
at what ratio does a detergent completely solubilize membrane lipids?
molar ratio of D:L of 2:1
89
at what ratio will a membrane become leaky, but not solubilized?
D:L = 1:2
90
what can be caused by using too much detergent?
- depletion of transmembrane domains-interacting lipids
- expose hydrophobic protein patches
- aggregate proteins
91
name one denaturing and one non-denaturing detergent?
SDS = denaturing
Triton X-100 = non-denaturing
