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ANAT458 > Analysis of membrane components - lect 3-5 > Flashcards

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

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2
Q

what are the 2 techniques used to find out analyse the TOPOLOGY of membrane proteins?

A
  • glycosylation mapping
  • natural/artificial epitope-tag
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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)

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4
Q

what residue (aa) get glycosylated?

A

N (Asparagine), R (arginine), S (serine), T (threonine), Y (tyrosine)

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5
Q

what is the procedure for glycosylation mapping?

A
  1. mutagenize (KO) putative (tentative) glycosylation sites
  2. determine which site is glycosylated by comparing the mutants vs WT
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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

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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

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8
Q

what is the procedure for using epitope tags?

A
  1. mutagenesis: insert an epitope in your protein which you have an antibody for
  2. express the protein in a cell line
  3. expose the cells to the antibody
  4. detect the signal by fluorescent microscopy or by FACS
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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
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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

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11
Q

name the methods used for protein localization

A
  • FRAP
  • Immunofluorescence and FACS
  • GFP-localization
  • Cell surface biotinylation
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12
Q

what is FRAP?

A

fluorescence recovery after photobleaching: method to study membrane dynamics in live cells

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13
Q

what is the procedure for FRAP?

A
  1. shine a high energy laser beam (bleach) on a well-defined spot in fluorescently-labelled cells
  2. Allow the fluorescence to recover and monitor the recovery time
  3. repeat in different conditions
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14
Q

what kind of proteins take more time to recover from bleaching?

A

proteins take longer than lipids

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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

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16
Q

what % of lipids are mobile?

A

100%, and they diffuse fast

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17
Q

what is the definition of immunofluorescence?

A

IF: Immuno-affinity method that allows to study component cellular localization by microscopy

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18
Q

what is FACS?

A

flow cytometry = fluorescence-activated cell sorting of immuno-labelled material

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19
Q

what are the first steps of immunofluorescence AND FACS?

A
  1. express your protein in a cell line (dead)
  2. incubate your cells with a fluorescently-tagged antibody for your protein
  3. counterstain other cell compartments with another fluorescent dye
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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

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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

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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
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23
Q

compared to IF, FACS does not give you information about what?

A

about cellular localization

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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

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25
Q

what is GFP?

A

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

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26
Q

how can you tag multiple proteins at a time?

A

by using analogues of GFP such as YFP and CFP that emit fluorescence at other wavelengths

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27
Q

what do you need to be careful for when using GFP?

A

that the fusion did not alter the protein’s function and/or cellular localization

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28
Q

what is cell-surface biotinylation?

A

Addition of a modified biotin molecule to cell surface proteins

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29
Q

what are the principles on which cell-surface biotinylation rely?

A
  • Sulfo-NHS-Biotin derivative does not cross the plasma membrane
  • Chemically modified biotin make it reactive to covalently bind to primary amines (NH2)
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30
Q

what is steptavidin? what is it used for?

A

a protein with very high affinity to biotin: can be used to pull down or analyze biotinylated proteins

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31
Q

what is required at cell surface for chemical reaction to primary amines (NH2 / mainly lysines) ?

A

free lysines

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32
Q

at what temperature does internalization stop?

A

4 deg celsius

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33
Q

sulfo-NHS-biotin and the protein of interest become ______ bound via primary _______

A

covalently;
amines (NH2)

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34
Q

what do you do after biotinylating your protein?

A

solubilise and precipitate the proteins

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35
Q

name different processes you can study via biotinylation

A

surface biotinylation, endocytosis biotinylation, re-insertion biotylation
(basically LOCALIZING the protein)

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36
Q

what is protein complementation assay?

A

Method of identification of protein-protein interactions based on the association of two proteins that generate a detectable readout

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37
Q

what are “bait” and “prey” in the protein complementation assay?

A

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
Q

name reporter proteins

A
  • DHFR
  • Beta-lactamase
  • eGFP
  • Horseradish peroxidase
  • Luciferase
39
Q

how does “survival selection” in the protein complementation assay work?

A

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
Q

explain the Protein Complementation Assays: Yeast 2 Hybrid system

A

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
Q

how do you confirm that the protein you found that interact with your protein of interest are not just artefacts?

A
  1. classify the preys based on their function
  2. verify by functional assay
42
Q

what is co-ip?

A

Co-precipitation of protein(s) that interact with your protein of interest

43
Q

what are the steps of co-ip?

A
  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
Q

what are limitations of co-ip?

A
  • 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
Q

what are the requirements for an interaction to be able to resist detergent solubilization?

A
  • interaction must occur outside a hydrophobic region
  • interaction must occur through polar forces
46
Q

what is a variant of co-ip for when there is no specific ab for out WT protein of interest?

A

using a mutant epitope-tagged protein of interest so it can bind our Ab

47
Q

what is cross-linking?

A

chemical linking reaction between two nearby proteins using a bis-functional chemical reagent

48
Q

describe a bis-functional reagent

A

2 reactive groups linked by a spacer arm

49
Q

what is cross-linking good to study?

A

to study weak or transient protein-protein interactions, because cross-linking stabilizes them

50
Q

the length of the spacer arm determines what?

A

the radius of action of th cross-linking agent

51
Q

how does the cross-linking reaction happens?

A

via the primary amines (lysine side chain) of the proteins of interest

52
Q

how can you study your cross-link once the reaction is done?

A

you can solubilize the proteins, immunoprecipitate, and analyze by western blot or mass spec.

53
Q

what characteristics can cross-linkers have?

A

can be impermeant (charged) or permeant (no charge);
can be cleavable or (s-s bond) or not cleavable

54
Q

what is the Bio-ID method?

A

Protein-protein interaction detection method using the fusion of a protein of interest with the biotin ligase BirA

55
Q

how does bio-ID work?

A

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
Q

what is tricky about Bio-ID?

A

it only labels proteins in the vicinity of your protein of interest and doesn’t guaranty that the proteins make direct contact

57
Q

what is FRET and BRET?

A

fluorescence / bioluminescence Resonance Energy Transfer

58
Q

what does FRET/BRET allow us to do?

A

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
Q

in what case with you see luminescence in FRET/BRET?

A

if proteins A and B interact, CFP will transfer energy to YFP (YFP absorbs the resonance energy from CFP and then releases it)

60
Q

what are the conditions for FRET/BRET to function?

A
  • The acceptor’s absorption spectrum must approximately match the donor’s fluorescence spectrum
  • the two molecules must be in very close proximity
61
Q

The efficiency of energy transfer in FRET is inversely proportional to what?

A

the 6th power of the distance

62
Q

what is the formula for FRET efficiency?

A

E = 1 / (1 + R/R0)^6 )

63
Q

what is R0? (in FRET)

A

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
Q

typically, the energy transfer of FRET works well in what radius?

A

R0/2 to 2R0 (usually 1-12 nm)

65
Q

what constants / values of protein interaction can you determine using FRET? how?

A

via ligand association assay:
- affinity constant (Ka)
- maximal binding (Bmax)
via ligand competition assay:
- dissociation constant (Kd)

66
Q

what is a ligand competition assay with FRET?

A

if there is competition between ligands, the FRET signal will decrease as we increase the competitor ligand concentration

67
Q

difference between FRET and BRET?

A

for bioluminescence, you dont need to shine light because the first molecule (Coelenterazine) is luminescent

68
Q

what is the crude method for LIPID analysis?

A

total extraction

69
Q

what does an organic solvent do?

A

separates the hydrophobic molecules (lipids) from the aqueous phase

70
Q

what is a TLC plate?

A

thin layer chromatography

71
Q

how does TLC work?

A

separate molecules based on their affinity for a solvent (aka how polar they are)

72
Q

how does liquid chromatography work?

A

measures the affinity of a protein / lipid to the liquid phase compared to the solid phase

72
Q

name the steps of total lipid extraction

A
  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
Q

what are the advantages and disadvantages of total lipid extraction?

A

Advantage: Cheap, rapid
Disadvantage:
- No information on organelles
- No information on asymmetry

74
Q

what do phospholipases do?

A

they cleave phospholipids on the outer leaflet of the bilayer (chops the phospho head group)

75
Q

how can you use phospholipases to study lipids composition?

A

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
Q

what is FITC-Annexin V used for?

A

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
Q

what are the steps of immunoprecipitation

A
  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
Q

what kind of detergent is SDS?

A

a denaturing detergent

79
Q

what method can you use to study a whole organelle?

A

sub-cellular fractionation

80
Q

why is 100% pure isolated membranes practically impossible to achieve?

A

because membrane disruption breaks up organelles, scrambles the 2 leaflets, causes fusion or adhesion of different membranes

81
Q

what do non-denaturing detergents do to cell membranes?

A

disrupts the membranes and takes the phospholipids away

82
Q

what can EDTA do?

A

prevents membranes from associating together (controls Ca2+ concentration)

83
Q

why are proteases inhibitors important when doing membrane isolation?

A

because proteins could get degraded by the lysosomal proteases released when lysosome gets broken in the centrifuge

84
Q

In absence of lipids, what happens to an amphiphilic peptide?

A

they aggregate together via hydrophobic patches when in a hydrophilic environment

85
Q

what’s the definition of a detergent?

A

Amphiphilic molecule which, when dissolved in water, is used to solubilize other molecules

86
Q

what is cmc?

A

critical micelle concentration: the saturating concentration of detergent monomer = the concentration of detergent at which micelles first appear

87
Q

why do detergents form micelles while phospholipids form bilayers?

A

because of their shape (triangular shape of detergent amphipathic molecules)

88
Q

at what ratio does a detergent completely solubilize membrane lipids?

A

molar ratio of D:L of 2:1

89
Q

at what ratio will a membrane become leaky, but not solubilized?

A

D:L = 1:2

90
Q

what can be caused by using too much detergent?

A
  • depletion of transmembrane domains-interacting lipids
  • expose hydrophobic protein patches
  • aggregate proteins
91
Q

name one denaturing and one non-denaturing detergent?

A

SDS = denaturing
Triton X-100 = non-denaturing

ANAT458 (10 decks)

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