Transcript Co-Localization Techniques - FSU Program in Neuroscience

Co-Localization of Proteins –
Standard Techniques
and
Biophysical Approaches
Debra Ann Fadool
Harianto Tjong
30 November 2007
Physiological Context for Protein Interaction
Studies and Co-localization
Estimated that there are ~500,000 proteins in
the human body and approximately 10,000 can
be produced by a single cell.
Estimated that 80% of ALL proteins DO NOT
exist in isolation, but rather exist in a protein
complex.
Protein-protein interactions are contained in
cellular networks known as “hubs” with
connecting “nodes”.
Gene-targeted deletion of a major node within a
given hub = “Central-lethality Rule”.
Protein-protein Interactions Fall within the
Study of Proteomics
Association of proteins in stable or transient
complexes, generally determine a linked
function in a cascade.
The binding of one signaling protein to another
can recruit proteins to a subcellular location.
Interactions between proteins can introduce
necessary conformational changes required for
protein activity.
Binary protein-protein interactions are the
cornerstone of signal transduction.
Higher-order assemblages or scaffolds provide
efficiency of the transmission of the signal.
How does one identify and characterize
protein-protein interactions?
Standard Techniques
Glutathione-S-Transferase Fusion Proteins
Affinity Tags
Tandem Affinity Purification (TAP) Tags
Strep-Tag III
Quantitative Proteomics
Chemical Crosslinking
Two-hybrid Yeast
Phage-display
Universal Verification of Interaction Techniques
Co-Immunoprecipitation
Confocal Microscopy
Public Protein-Protein Interaction Databases
Biophysical Verification of Interaction Techniques
Fluorescence Resonance Energy Transfer (FRET)
GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)
Mass Spectroscopy (MS)
Atomic Force Microscopy (AFM)
Surface Plasmon Resonance (SPR)
Standard Techniques
Glutathione-S-Transferase Fushion Proteins (GST-Pull Down Assays)
1. GST-fusion protein is expressed and affinity
purified in bacteria. This is your bait protein.
2. Then mix your GST-fusion protein with cell
lysate to bind interaction partner.
3. Purify interacting protein using GST beads
or a SwellGel Disc.
4. Run samples out on SDS-PAGE
5. Good for interactions in solution
6. Far Western Analysis –
Use GST 32P labeled fusion protein and then
cleave off the GST.
Probe an expression library or SDS-Page
nitrocellulose and scan for radioactive band.
7. Limitation: Completely in vitro
Standard Techniques
Affinity Tags for Purification of Protein Complexes
1. This is a genetically-fused affinity tag.
2. Cells are transfected with the plasmid
containing the bait protein and the tag.
3. The bait protein and associated partners
are then isolated using a solid support for the
specific tag.
4. Advantage: can be greater than binary.
5. Advantage: can have post-translational
modifications that can increase complexes
(such as phosphorylation).
6. Disadvantage: non-physiological targets
can combine or two proteins from different
cells or subcellular compartments.
7. Concept of: “Macromolecular Crowding”
Standard Techniques
Affinity Tags for Purification of Protein Complexes
8. Disadvantage: can be highly biased
toward proteins of high abundance in a cell.
9. Disadvantage: the tag might even affect
the subcellular localization in a cell or its
protein partner.
10. Solution? = Put tags in different places
within the bait protein.
11. Solution? = Use Two Sequential
Affinity Tags not One….
This is called TAP- method
Standard Techniques
Tandem Affinity Purification (TAP) Method
12. There is a TEV cleavage site between the
tandem tags.
13. After binding to putative protein partner,
run over a column using protein A (first tag),
then cleave, and run over a second column
using CHP/SBP/Strep II (second tag) Thus you have a double purifcation.
14. Disadvantages: need large starting material
for double purification and cannot capture
transient protein partners.
Standard Techniques
Strep-tag III
15. These are the most commonly used
and commercially available tags packaged
in kits with the antisera.
16. Small peptide tags = called epitope tagging
Flag-tag, Myc-tag, Lap-tag, or HA-tag.
17. Advantage: Small tag is less likely to
Interfere with binding of protein partner.
18. Disadvantage: Is good for even low-afinity
binding targets but this can also increase
non-specific background.
Using Epitope Tags to Look at Membrane Localization
Untagged Channel
Routine Immunoprecipitation (IP)
and C-IP; Native and Cloned
N-terminal GFP Tagged Channel
Total Channel Expression
S1/S2 Extracellular Myc
Tagged Channel
Surface Channel Expression
How Do Tags Reveal Protein Subcellular
Localization?
Total Channel Expression
Kv1.3
Kv1.3
TrkB
Kv1.3
TrkB
nShc
Surface Channel Expression
Kv1.3
Kv1.3
TrkB
Kv1.3
TrkB
nShc
Imaging channel localization using
multiple tags (Kv1.3-myc-GFP)
c-myc: cell surface
GFP: total channel
combined image
(DAPI: nucleus)
Standard Techniques
Quantitative Proteomics
1. Culture cells in media supplemented with labelled amino acids
or radioactive probes.
2. Differentially label proteins with
metabolic or isotope tags.
3. Non-specific vs. specific protein
partners can be distinquished based
upon intensity of the peaks
observed under MS.
A = SILAC = Metabolic labelling technique
B = ICAT = Isotope-coded affinity tag
Standard Techniques
Chemical Crosslinking
1. Oldest technique to study protein-protein
Interactions (> 50 years).
2. Completely empirical: never know which
proteins can be crosslinked and by which
chemical reagents!
3. “The absence of evidence is not
necessarily evidence of absence.”
4. Some linkers are photoactivatable so can
study kinetics of dynamic interactions.
5. Can also determine distance between
protein partners dependent upon the chemistry
of the linker (zero-length cross-linkers; molecular
rulers, and nearest neighbors).
6. After crosslink, analysis by SDS-PAGE,
peptide mapping by HPLC, MS, or affinity
purifcation.
Standard Techniques
Yeast Two-Hybrid
BD = binding to promoter DNA sequence
AD = binding to DNA that activates transcription
When bait and partner bind, then creates
intact and functional transcription regulator
to induce production of reporter gene product.
1. Advantage = Unknown target partners
can be discovered via “fishing”.
2. Advantage = Transient and weak
interactions can be detected using the
genetic reporter system.
3. Disadvantage = 50% False Positives!
4. Disadvantage = only binary interactions
5. Yeast does not = Mammal
Must Validate all Protein-protein
Interactions by Multiple Methods!
Due to false positives, only 10% of the
entire human interaction maps are complete!
Types of Validation Approaches:
1. Confocal Microscopy
2. Co-Immunoprecipitation
3. Surface Plasmon Resonance
4. Spectroscopy
Verification of Interaction
Techniques
A. Confocal Microscopy for Subcellular
Co-localization
1. Core facility equipment.
2. Can be native or
cloned protein partners.
Verification of Interaction
Techniques
B. Reciprocal Co-Immunoprecipitation
1. Incubate with antiserum directed
against bait protein, SDS-PAGE,
then probe with partner antiserum.
2. Repeat in opposite direction.
3. Advantage = Gold Standard
4. Disadvantage = Must have
antisera source to proteins of interest
Protein-protein Interaction
Databases
http://www.piercenet.com/Products/Browse.cfm?fldID=A6C041924535-4618-B372-98D97A7A21F8 (Great Technical Resource)
iHOP http://www.ihop-net.org/UniPUb/iHOP/
IntAct http://www.ebi.ac.uk/intact
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P1538
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EBI63147
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P78352,
EBI80389
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physical interaction
physical interaction
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EBI-631396
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Biophysical Approaches
C. Surface Plasmon Resonance (SPR)
1. Calculation of the microscopic rate
constants for interaction between proteins.
2. Doesn’t have to be p-p but can be proteinnucleic acid, protein-ligand, or proteinnanoparticles.
3. One interaction partner (Y) is bound to the
metal film while the other (red balls) partner
Is injected over the surface.
4. Amount of bound protein is quantified
based upon angle of reflected light.
5. Real time association and dissociation
of a protein-protein interaction can be
quantified.
Biophysical Approaches
Surface Plasmon Resonance (SPR)
Plus Ca2+
No Ca2+
2 Control,
Non-interacting
Proteins
Biophysical Approaches
Surface Plasmon Resonance (SPR)
High-Throughput to Determine Multiple Ligand Interacting Molecules
Biophysical Approaches
D. Atomic Force Microscopy
Most Used for
Receptor-Ligand Interactions
1. Rather than binding affinities or rate constants
of association, will directly measure the forces
involved in creating and maintaining a p-p
Interaction.
2. Can be used to measure dynamic strength
and characterize free energy released during
breakage of a p-p interaction complex.
3. Uses a cantilever that flexibly bends according
to the topographical contour of the specimen
scanned.
4. Atomic-level resolution is reached by
translating the deflection of the cantilever into
an image map of surface height differences.
5. Then perform computer reconstruction to achieve
final image.
6. How we know that Streptavidin-biotin strongest
protein-protein interaction to date (300 piconewtons)
Biophysical Approaches
How does one identify and characterize
protein-protein interactions?
Standard Techniques
Glutathione-S-Transferase Fusion Proteins
Affinity Tags
Tandem Affinity Purification (TAP) Tags
Strep-Tag III
Quantitative Proteomics
Chemical Crosslinking
Two-hybrid Yeast
Phage-display
Universal Verification of Interaction Techniques
Co-Immunoprecipitation
Confocal Microscopy
Public Protein-Protein Interaction Databases
Biophysical Verification of Interaction Techniques
Fluorescence Resonance Energy Transfer (FRET)
GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)
Mass Spectroscopy (MS)
Atomic Force Microscopy (AFM)
Surface Plasmon Resonance (SPR)
FRET
What is FRET?
How it works
What is it for?
Example
Förster/Fluorescent Resonance
Energy Transfer
Fluorescence:
a luminescence phenomenon in which the molecular absorption of a
photon triggers (almost spontaneously) the emission of another
photon with a longer wavelength.
The energy difference of these photons are converted to molecular
vibrations or heat.
Quantum yield = (# of photons emitted) / (# of photons absorbed)
Lifetime refers to the average time the molecule stays in its exited
state before emitting a photon.
Energy Transfer involves 2 different fluorescent molecules
(fluorophores): donor (D) and acceptor (A)
The key is non-radiative
FRET: a non-radiative, dipole-dipole coupling process, transfer
energy from an excited donor fluorophore to an acceptor
fluorophore in very close proximity (typically within 10nm)
Nat. Rev. Mol. Cell Biol. 4,
579 (2003)
The Förster theory
Förster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz.
Annalen der Physik 2, 55-75.
The rate at which Förster energy transfer occurs is given
by:
6
 R0 
kD  1  D
kt  k D  
 r 
Efficiency varies as the sixth
power of the distance
between D – A
EFRET 
kt
1

kt  k D 1  (r / R0 )6
The Förster theory
Förster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz.
Annalen der Physik 2, 55-75.
Förster distance, R0, is the distance at which 50% energy transfer takes
place, depends on
• quantum yield (Q) of the donor
• relative orientation of the transition dipoles of the two fluorophores, 2.
• acceptor extinction coefficient, e.
• Spectral overlap between D emission and A absorption, J.
• Refractive index of medium, n, usually for water solvent: 1.4

R0  970  2 J ( )n  4Q
J   


0

1/ 6
nm
F    e    4d

 F  d
0
Example: the emission and absorption spectra of cyan fluorescent protein (CFP,
the donor) and yellow fluorescent protein (YFP, the acceptor), respectively.
CFP & YFP pair is currently the ‘best’ for FP-based FRET.

R0  970  2 J ( )n  4Q

1/ 6
nm
The theory supporting energy transfer is based on the concept of treating
an excited fluorophore as an oscillating dipole that can undergo an energy
exchange with a second dipole having a similar resonance frequency
K2 is the degree of alignment: D emission dipole
& A absorption dipole, ranging from 0 to 4
• maximum if the two dipoles are both parallel
and collinear
• 0 if perpendicular
• 2/3 is usually assumed, which is the average
value integrated over all possible angles
• because of the sixth-root relationship to the
Förster distance, a variation from 1-4 produces
~26% change

R0  970  2 J ( )n  4Q

1/ 6
nm
Donor
Acceptor
Förster Distance
(Nanometers)
Tryptophan
Dansyl
2.1
IAEDANS (1)
DDPM (2)
2.5 - 2.9
BFP
DsRFP
3.1 - 3.3
Dansyl
FITC
3.3 - 4.1
Dansyl
Octadecylrhodamine
4.3
CFP
GFP
4.7 - 4.9
CF (3)
Texas Red
5.1
Fluorescein
Tetramethylrhodamine
4.9 - 5.5
Cy3
Cy5
>5.0
GFP
YFP
5.5 - 5.7
BODIPY FL (4)
BODIPY FL (4)
5.7
Rhodamine 6G
Malachite Green
6.1
FITC
Eosin Thiosemicarbazide
6.1 - 6.4
B-Phycoerythrin
Cy5
7.2
Cy5
Cy5.5
>8.0
(1) 5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid
(2) N-(4-dimethylamino-3,5-dinitrophenyl)maleimide
(3) carboxyfluorescein succinimidyl ester
(4) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
http://www.olympusfluoview.com/applications/fretintro.html
Molecular Ruler
EFRET
16
 1

r  R0 
 1
 EFRET 
kt
1


kt  k D 1  (r / R0 )6
EFRET
D
 1
 DA
Thanks to its high resolution, FRET has been used to
measure protein-protein interactions between 2 FPlabeled proteins.
Biosensors
FRET is well-suited
to study protein
interactions.
FRET also can be useful to
study kinetics of
association/dissociation
between macromolecules
Biosensors
Problems
The D and A fluorophores might be different brightness.
D:A stoichiometry is outside the range of 1:10 – 10:1
Cross-talk or bleed-through between the 2 fluorophore colors:
– Direct excitation of A.
– D emits fluorescence that leaks into detection channel for A
fluorescence.
Problems
FRET signals reduces if D & A are not aligned
or not within R0
Structure of GFP which
occupies much of the
useful FRET distance.
Simultaneous optical and electrical recording of
single gramicidin channels.
Biophys. J. 84, 612 (2003)
Central aim: to test the
feasibility of combined
optical and electrical
recording measurements in
a configuration that could
prove applicable to the
study of a wide range of ion
channels.
System: Gramicidin A
Has served as a model system for understanding
fundamental aspects of ion channels for more than 30
years.
Was the first channel for which a primary structure was
determined.
Was the first defined substance of which single-channel
currents were observed via electrical recording.
Was the 1st channel for which the 3D structure of the
conducting form was determined.
The gating event (channel opening) is widely believed to
be the dimerization of peptides in the membrane.
Dimerization results in a pathway
for ions
Due to the symmetric composition of both membrane leaflets, dimers
can be Cy3/Cy3 or Cy5/Cy5 or Cy3/Cy5
FRET is expected to occur only
with a current of 1.65 – 1.75 pA at
100mV
Uncorrelated electrical and
optical events
FRET signals observed even when non-ion
channels were detected electrically.
Possibilities:
• dimer formed in a non-bilayer part
• occurrence of the low-conductance double-stranded dimers.
• non-conducting dimeric intermediate was observed. ->
Advantage of single molecule imaging.
Uncorrelated electrical and
optical events
Electrical signals were observed without FRET.
Possibilities:
• dimer formed outside the imaging area
• photo-destruction of the donor without affecting the conductance
properties of the channel.
• inefficient FRET because some dyes were not freely rotating, or to
far from optimum distance.
Trajectories from red spots: diffusion
coefficient estimation for the dimeric channel
Thanks!