Co-Localization of Proteins –Co-Localization of Proteins –

Standard TechniquesStandard Techniquesandand

Biophysical ApproachesBiophysical Approaches

Debra Ann FadoolDebra Ann Fadool

Harianto TjongHarianto Tjong

30 November 200730 November 2007

Physiological Context for Protein Interaction Physiological Context for Protein Interaction Studies and Co-localizationStudies and Co-localization

Estimated that there are ~500,000 proteins in Estimated that there are ~500,000 proteins in the human body and approximately 10,000 can the human body and approximately 10,000 can be produced by a single cell.be produced by a single cell.Estimated that 80% of ALL proteins DO NOT Estimated that 80% of ALL proteins DO NOT exist in isolation, but rather exist in a protein exist in isolation, but rather exist in a protein complex.complex.Protein-protein interactions are contained in Protein-protein interactions are contained in cellular networks known as “hubs” with cellular networks known as “hubs” with connecting “nodes”. connecting “nodes”. Gene-targeted deletion of a major node within a Gene-targeted deletion of a major node within a given hub = “Central-lethality Rule”.given hub = “Central-lethality Rule”.

Protein-protein Interactions Fall within the Protein-protein Interactions Fall within the Study of ProteomicsStudy of Proteomics

Association of proteins in stable or transient Association of proteins in stable or transient complexes, generally determine a linked function complexes, generally determine a linked function in a cascade.in a cascade.The binding of one signaling protein to another The binding of one signaling protein to another can recruit proteins to a subcellular location.can recruit proteins to a subcellular location.Interactions between proteins can introduce Interactions between proteins can introduce necessary conformational changes required for necessary conformational changes required for protein activity.protein activity.Binary protein-protein interactions are the Binary protein-protein interactions are the cornerstone of signal transduction.cornerstone of signal transduction.Higher-order assemblages or scaffolds provide Higher-order assemblages or scaffolds provide efficiency of the transmission of the signal.efficiency of the transmission of the signal.

How does one identify and characterize How does one identify and characterize protein-protein interactions?protein-protein interactions?

Standard TechniquesStandard TechniquesGlutathione-S-Transferase Fusion ProteinsGlutathione-S-Transferase Fusion ProteinsAffinity TagsAffinity TagsTandem Affinity Purification (TAP) TagsTandem Affinity Purification (TAP) TagsStrep-Tag IIIStrep-Tag IIIQuantitative ProteomicsQuantitative ProteomicsChemical CrosslinkingChemical CrosslinkingTwo-hybrid YeastTwo-hybrid YeastPhage-display Phage-display

Universal Verification of Interaction TechniquesUniversal Verification of Interaction TechniquesCo-ImmunoprecipitationCo-ImmunoprecipitationConfocal MicroscopyConfocal Microscopy

Public Protein-Protein Interaction DatabasesPublic Protein-Protein Interaction Databases

Biophysical Verification of Interaction TechniquesBiophysical Verification of Interaction TechniquesFluorescence Resonance Energy Transfer (FRET)Fluorescence Resonance Energy Transfer (FRET)GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)Mass Spectroscopy (MS)Mass Spectroscopy (MS)Atomic Force Microscopy (AFM)Atomic Force Microscopy (AFM)Surface Plasmon Resonance (SPR)Surface Plasmon Resonance (SPR)

Standard TechniquesStandard Techniques

Glutathione-S-Transferase Fushion Proteins (GST-Pull Down Assays)Glutathione-S-Transferase Fushion Proteins (GST-Pull Down Assays)

1. GST-fusion protein is expressed and affinitypurified 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 beadsor a SwellGel Disc.4. Run samples out on SDS-PAGE5. Good for interactions in solution6. Far Western Analysis –

Use GST 32P labeled fusion protein and thencleave off the GST.Probe an expression library or SDS-Pagenitrocellulose and scan for radioactive band.

7. Limitation: Completely in vitro

Standard TechniquesStandard Techniques

Affinity Tags for Purification of Protein ComplexesAffinity Tags for Purification of Protein Complexes

1. This is a genetically-fused affinity tag.2. Cells are transfected with the plasmidcontaining the bait protein and the tag.3. The bait protein and associated partnersare then isolated using a solid support for the specific tag.4. Advantage: can be greater than binary.5. Advantage: can have post-translationalmodifications that can increase complexes(such as phosphorylation).6. Disadvantage: non-physiological targetscan combine or two proteins from differentcells or subcellular compartments.7. Concept of: “Macromolecular Crowding”

Standard TechniquesStandard Techniques

Affinity Tags for Purification of Protein ComplexesAffinity 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 affectthe subcellular localization in a cell or itsprotein partner.10. Solution? = Put tags in different placeswithin the bait protein.11.Solution? = Use Two Sequential Affinity Tags not One….

This is called TAP- method

Standard TechniquesStandard Techniques

Tandem Affinity Purification (TAP) MethodTandem 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 columnusing CHP/SBP/Strep II (second tag) -Thus you have a double purifcation.

14.Disadvantages: need large starting materialfor double purification and cannot capture transient protein partners.

Standard TechniquesStandard Techniques

Strep-tag IIIStrep-tag III

15.These are the most commonly usedand commercially available tags packagedin 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-afinitybinding targets but this can also increasenon-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 MycTagged Channel

Surface Channel Expression

How Do Tags Reveal Protein SubcellularLocalization?

Total Channel Expression

Surface Channel Expression

Kv1.3 Kv1.3TrkB

Kv1.3TrkBnShc

Kv1.3 Kv1.3TrkB

Kv1.3TrkBnShc

Imaging channel localization using Imaging channel localization using

multiple tags (Kv1.3-myc-GFP)multiple tags (Kv1.3-myc-GFP)

c-myc: cell surface GFP: total channel combined image(DAPI: nucleus)

Standard TechniquesStandard Techniques

Quantitative ProteomicsQuantitative Proteomics

A = SILAC = Metabolic labelling technique

B = ICAT = Isotope-coded affinity tag

1. Culture cells in media supple-mented with labelled amino acidsor radioactive probes.2. Differentially label proteins with metabolic or isotope tags.3. Non-specific vs. specific proteinpartners can be distinquished basedupon intensity of the peaks observed under MS.

Standard TechniquesStandard Techniques

Chemical CrosslinkingChemical Crosslinking1. Oldest technique to study protein-proteinInteractions (> 50 years).2. Completely empirical: never know whichproteins 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 betweenprotein partners dependent upon the chemistryof the linker (zero-length cross-linkers; molecular rulers, and nearest neighbors).6. After crosslink, analysis by SDS-PAGE, peptide mapping by HPLC, MS, or affinitypurifcation.

Standard TechniquesStandard Techniques

Yeast Two-HybridYeast Two-HybridBD = binding to promoter DNA sequenceAD = binding to DNA that activates transcription

When bait and partner bind, then createsintact and functional transcription regulatorto 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 interactions5. Yeast does not = Mammal

Must Validate all Protein-protein Must Validate all Protein-protein Interactions by Multiple Methods!Interactions by Multiple Methods!

Due to false positives, only 10% of theDue to false positives, only 10% of theentire human interaction maps are complete!entire human interaction maps are complete!

Types of Validation Approaches:Types of Validation Approaches:1.1. Confocal MicroscopyConfocal Microscopy2.2. Co-ImmunoprecipitationCo-Immunoprecipitation3.3. Surface Plasmon ResonanceSurface Plasmon Resonance4.4. SpectroscopySpectroscopy

Verification of Interaction Verification of Interaction TechniquesTechniques

A. Confocal Microscopy for Subcellular Co-localization

1. Core facility equipment.2. Can be native or cloned protein partners.

Verification of Interaction Verification of Interaction TechniquesTechniques

B. Reciprocal Co-ImmunoprecipitationB. Reciprocal Co-Immunoprecipitation1. Incubate with antiserum directedagainst bait protein, SDS-PAGE, then probe with partner antiserum.2. Repeat in opposite direction.3. Advantage = Gold Standard4. Disadvantage = Must have antisera source to proteins of interest

Protein-protein Interaction Protein-protein Interaction DatabasesDatabases

http://www.piercenet.com/Products/Browse.cfm?fldID=A6C04192-4http://www.piercenet.com/Products/Browse.cfm?fldID=A6C04192-4535-4618-B372-98D97A7A21F8535-4618-B372-98D97A7A21F8 (Great Technical Resource) (Great Technical Resource)

iHOP iHOP http://www.ihop-net.org/UniPUb/iHOP/http://www.ihop-net.org/UniPUb/iHOP/

IntAct IntAct http://www.ebi.ac.uk/intacthttp://www.ebi.ac.uk/intact

1

P15384, EBI-631478

P78352, EBI-80389

PSD95

Kcna3

DLG4

10116(rat)

9606(human)

Kim et al. (1995) Kim et al. (1995)

7477295 7477295

physical interaction physical interaction

2 hybrid far western blotting

intact intact

EBI-631396 EBI-631316

Biophysical ApproachesBiophysical Approaches

C. Surface Plasmon Resonance (SPR)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 protein-nucleic acid, protein-ligand, or protein-nanoparticles.3. One interaction partner (Y) is bound to the metal film while the other (red balls) partnerIs injected over the surface.4. Amount of bound protein is quantifiedbased upon angle of reflected light.5. Real time association and dissociationof a protein-protein interaction can bequantified.

Biophysical ApproachesBiophysical Approaches

Surface Plasmon Resonance (SPR)Surface Plasmon Resonance (SPR)

Plus Ca2+

No Ca2+

2 Control,Non-interactingProteins

Biophysical ApproachesBiophysical Approaches

Surface Plasmon Resonance (SPR)Surface Plasmon Resonance (SPR)

High-Throughput to Determine Multiple Ligand Interacting Molecules

Biophysical ApproachesBiophysical Approaches

D. Atomic Force MicroscopyD. Atomic Force Microscopy

1. Rather than binding affinities or rate constantsof association, will directly measure the forcesinvolved in creating and maintaining a p-pInteraction.2. Can be used to measure dynamic strengthand characterize free energy released duringbreakage of a p-p interaction complex.3. Uses a cantilever that flexibly bends accordingto the topographical contour of the specimen scanned.4. Atomic-level resolution is reached by translating the deflection of the cantilever intoan image map of surface height differences.5. Then perform computer reconstruction to achievefinal image. 6. How we know that Streptavidin-biotin strongestprotein-protein interaction to date (300 piconewtons)

Most Used for Receptor-Ligand Interactions

Biophysical ApproachesBiophysical Approaches

How does one identify and characterize How does one identify and characterize protein-protein interactions?protein-protein interactions?

Standard TechniquesStandard TechniquesGlutathione-S-Transferase Fusion ProteinsGlutathione-S-Transferase Fusion ProteinsAffinity TagsAffinity TagsTandem Affinity Purification (TAP) TagsTandem Affinity Purification (TAP) TagsStrep-Tag IIIStrep-Tag IIIQuantitative ProteomicsQuantitative ProteomicsChemical CrosslinkingChemical CrosslinkingTwo-hybrid YeastTwo-hybrid YeastPhage-display Phage-display

Universal Verification of Interaction TechniquesUniversal Verification of Interaction TechniquesCo-ImmunoprecipitationCo-ImmunoprecipitationConfocal MicroscopyConfocal Microscopy

Public Protein-Protein Interaction DatabasesPublic Protein-Protein Interaction Databases

Biophysical Verification of Interaction TechniquesBiophysical Verification of Interaction TechniquesFluorescence Resonance Energy Transfer (FRET)Fluorescence Resonance Energy Transfer (FRET)GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)GFP-Protein Proximity Imaging Microscopy (GFP-PRIM)Mass Spectroscopy (MS)Mass Spectroscopy (MS)Atomic Force Microscopy (AFM)Atomic Force Microscopy (AFM)Surface Plasmon Resonance (SPR)Surface Plasmon Resonance (SPR)

FRETFRET

What is FRET?What is FRET?

How it worksHow it works

What is it for?What is it for?

ExampleExample

FFöörster/Fluorescent Resonance rster/Fluorescent Resonance Energy TransferEnergy Transfer

Fluorescence:Fluorescence:

a luminescence phenomenon in which the molecular absorption of a a luminescence phenomenon in which the molecular absorption of a photon triggers (almost spontaneously) the emission of another photon triggers (almost spontaneously) the emission of another photon with a longer wavelength.photon with a longer wavelength.

The energy difference of these photons are converted to molecular The energy difference of these photons are converted to molecular vibrations or heat.vibrations or heat.

Quantum yield = (# of photons emitted) / (# of photons absorbed)Quantum yield = (# of photons emitted) / (# of photons absorbed)

Lifetime refers to the average time the molecule stays in its exited Lifetime refers to the average time the molecule stays in its exited state before emitting a photon.state before emitting a photon.

Energy Transfer involves 2 different fluorescent molecules (fluorophores): donor (D) and acceptor (A)

The key is non-radiativeThe key is non-radiativeFRET: a non-radiative, dipole-dipole coupling process, transfer FRET: a non-radiative, dipole-dipole coupling process, transfer energy from an excited donor fluorophore to an acceptor energy from an excited donor fluorophore to an acceptor fluorophore in very close proximity (typically within 10nm)fluorophore in very close proximity (typically within 10nm)

Nat. Rev. Mol. Cell Biol. 4,

579 (2003)

The FThe Föörster theoryrster theoryFFöörster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz. rster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz.

Annalen der PhysikAnnalen der Physik 2, 55-75. 2, 55-75.

Efficiency varies as the sixth Efficiency varies as the sixth power of the distance between power of the distance between D – AD – A

60 )/(1

1

Rrkk

kE

Dt

tFRET

The rate at which FThe rate at which Föörster energy transfer occurs is given rster energy transfer occurs is given by:by:

6

0

r

Rkk Dt DDk 1

The FThe Föörster theoryrster theoryFFöörster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz. rster, T. (1948) Zwichenmolekulare energiewanderung und fluoreszenz.

Annalen der PhysikAnnalen der Physik 2, 55-75. 2, 55-75.

nm)(9706/142

0 QnJR

FFöörster distance, Rrster distance, R00, is the distance at which 50% energy transfer takes , is the distance at which 50% energy transfer takes place, depends onplace, depends on

• quantum yield (Q) of the donor

• relative orientation of the transition dipoles of the two fluorophores, 2.

• acceptor extinction coefficient, .

• Spectral overlap between D emission and A absorption, J.

• Refractive index of medium, n, usually for water solvent: 1.4

0

0

4

d

d

F

FJ

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.

nm)(9706/142

0 QnJR

2 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

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

nm)(9706/142

0 QnJR

Donor AcceptorFörster 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 RulerMolecular Ruler

61

0 11

FRETERr

DA

DFRETE

1

60 )/(1

1

Rrkk

kE

Dt

tFRET

Thanks to its high resolution, FRET has been used to Thanks to its high resolution, FRET has been used to measure protein-protein interactions between 2 FP-measure protein-protein interactions between 2 FP-labeled proteins.labeled proteins.

BiosensorsBiosensors

FRET also can be useful to FRET also can be useful to study kinetics of study kinetics of association/dissociation association/dissociation between macromoleculesbetween macromolecules

FRET is well-suited FRET is well-suited to study protein to study protein interactions.interactions.

BiosensorsBiosensors

ProblemsProblemsThe D and A fluorophores might be different brightness.The D and A fluorophores might be different brightness.

D:A stoichiometry is outside the range of 1:10 – 10:1D:A stoichiometry is outside the range of 1:10 – 10:1

Cross-talk or bleed-through between the 2 fluorophore colors:Cross-talk or bleed-through between the 2 fluorophore colors:– Direct excitation of A.Direct excitation of A.

– D emits fluorescence that leaks into detection channel for A D emits fluorescence that leaks into detection channel for A fluorescence.fluorescence.

ProblemsProblems

FRET signals reduces if D & A are not aligned FRET signals reduces if D & A are not aligned

or not within or not within RR00

Structure of GFP which occupies much of the useful FRET distance.

Simultaneous optical and electrical recording of Simultaneous optical and electrical recording of single gramicidin channels.single gramicidin channels.

Biophys. JBiophys. J. . 8484, 612 (2003), 612 (2003)

Central aim: to test the Central aim: to test the feasibility of combined feasibility of combined optical and electrical optical and electrical recording measurements in recording measurements in a configuration that could a configuration that could prove applicable to the prove applicable to the study of a wide range of ion study of a wide range of ion channels.channels.

System: Gramicidin ASystem: Gramicidin A

Has served as a model system for understanding Has served as a model system for understanding fundamental aspects of ion channels for more than 30 fundamental aspects of ion channels for more than 30 years.years.Was the first channel for which a primary structure was Was the first channel for which a primary structure was determined.determined.Was the first defined substance of which single-channel Was the first defined substance of which single-channel currents were observed via electrical recording.currents were observed via electrical recording.Was the 1Was the 1stst channel for which the 3D structure of the channel for which the 3D structure of the conducting form was determined.conducting form was determined.The gating event (channel opening) is widely believed to The gating event (channel opening) is widely believed to be the dimerization of peptides in the membrane.be the dimerization of peptides in the membrane.

Dimerization results in a pathway Dimerization results in a pathway for ionsfor 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

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 Uncorrelated electrical and optical eventsoptical 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.

Uncorrelated electrical and Uncorrelated electrical and optical eventsoptical events

Trajectories from red spots: diffusion coefficient estimation for the dimeric channel

Thanks!