Analyses for Molecular Interactions in Living Cells

Chi-Wu Chiang, Ph.D.Institute of Molecular Medicine

College of MedicineNational Cheng Kung University

Adopted from 2003-2004 catalog, Cell Signaling Technology, Inc.

Can these signaling networks be observed in living cells?

Yeast two-hybrid assay

Mammalian two-hybrid assay

Co-immunoprecipitation

Affinity purification

Co-localization by immunostaining

Traditional methods to detect protein-protein interactions

Seeing is believing

Fluorescent proteins

Green fluorescent protein (GFP)

Aequorea fluorescent protein (AFP) variants

Enhanced green fluorescent protein (EGFP)

Yellow fluorescent protein (YFP)

Enhanced yellow fluorescent protein (EYFP)

Cyan fluorescent protein (CFP)

Enhanced cyan fluorescent protein (ECFP)

From jellyfish Aequorea victoria

Fluorescent probes

Red fluorescent protein (RFP)

DsRED, DsRED2, DsRED-express, mRFP1

from Discosoma genus, Reef coral

Tended to be tetrameric

DsRED-monomer is a new RFP (45 amino acid substitutions of DsRED) without property of forming tetramer

ProteinExcitation Max nm

Emission Max nm

ECFP

EGFP

EYFPDsRED2

439

484

512

563

476

510

529

592

Excitation and Emission Spectra of Fluorescent Proteins

Ser65, Tyr66, and Gly67 are key residues to form chromophore

The structure of GFPGFP is an 11-stranded -barrel threaded by an -helix running up the axis of the cylinder .The chromophore is attached to the -helix and is buried almost perfectly in the center of the cylinder, which has been called a-can

Creation of monomeric fluorescent probes by mutagenesis

Interface disrupting mutation

Compare the wtGFP to EGFP

Increase in stability and brightness

Mutations in several residues, such as Ser65, Ala206, Leu221, Phe223

Dimerization at high concentrations was overcome

Compare EYFP to EGFP

YFP was rationally designed on the basis of the GFP crystal structure to red-shift the absorbance and emission spectra with respect to EGFP and other green fluorescent variants

YFP is much brighter than EGFP but is more sensitive to low pH and high halide concentrations

Factors affect the efficiency using the Fluorescence probes

Photostability-----caused by photobleaching

PH sensitivity-----most of the first-generation probes are acid sensitive

Oligomerizing property----using AFPs with Ala206Lys mutation

----using newest DsRED varients, such as mRFP1

Perturbation of intracellular conditions----introduction of the fluorescent probes may perturb the cellular

component of interest

Finding more fluorescent probes

From Renilla mulleriRenilla mulleri GFP, with narrow excitation and emission spectrua

From Anemonia sulcatadsFP593

More mutated variants

Small molecule probes

The biarsenical-tetracysteine system

CCPGCC

Nat. Rev. Mol. Cell Bio.3, 906-918 (2002).

Membrane-permeant fluorescein derative with two As substitutents, named FIAsH

Interaction of a single arsenic with a pair of thio groups is well known

Based on:

1,2-ethanedithiol (EDT) as a 1,2-dithiol antidotes to prevent non-specific labeling in cells

The biarsenical-tetracysteine system

CHoXAsH

Analogues of FIAsH have different excitation and emission spectra

FIAsH ReAsH

Current Methods in detecting protein-protein interactions in living cells

Fluorescence resonance energy transfer (FRET)

Bioluminescence resonance energy transfer (BRET)

Biomolecular luminescence/fluorescence complementation

-galactosidase/Luciferase complementation

Fluorescent protein complementation

Fluorescence (or Forster) Resonance Energy Transfer

FRET

Energy transfer between two fluorophores within distance on nanometer scales

FRET is the radiationless transfer of excited-state energy from an initially excited donor to an acceptor

Emission Absorption

The orientation factor, κ2, is given by κ2= (cosθT − 3cosθdcosθa)

Factors impact the rate of FRET

Proper spectral overlap of the donor and acceptor

r6

r, the distance between the two fluorophores

FRET is inversely proportional the distance between the fluorophores

< 10 nm or < 80 A aparto

Small fluorescent chemicals Larger fluorescent proteins

Compare a small fluorescent molecule-tagged FRET to a large fluorescent

molecule-tagged FRET

Free orientation and FRET is only limited by the distance factor

Limited orientation and spatially restricted, however, FRET is sensitive to orientation, distance, and conformation of two interacting molecules

The best pair of fluorophores are CFP and YFP

Basic designs for analysis of molecular interactions by FRET

CFP

YFP

Applications for monitoring molecular interactions in Living cells

a. Intermolecular FRET-based indicatorsG protein subunits dissociationTranscription factor homo- and heterodimerizationRas and Rap1 activation

b. Intramolecular FRET-based indicatorsCaspase activationCalcium flux sensorKinase activation

Applications for monitoring molecular interactions in Living cells

Monitor protein-protein interactionMonitor intramolecular conformational change

FRET applications

Calcium sensor (Cameleon)

M13, a peptide binds to calmodulin in calcium-dependent manner

Kinase activation sensor

Methods for FRET analysis in Living cells

Fluorescence Spectrophotometry

Spectrofluorimeter measurement

Cells, treated or not treated, suspensionIn PBS

Analyses by

Featured withExcitation light source, arc lamp, in UV or visible

Emission detector, such as photon counter or charged coupled device (CCD)

Scan full spectrum periodically, using filter sets and crystal counter< 1 second

Spectrofluorimeter measurement

FRET1433ECFP/1433EYFP

0

20000

40000

60000

80000

100000

120000

140000

160000

460

468

476

484

492

500

508

516

524

532

540

548

Wavelength

Em

issi

on in

tensi

ty

1433ECFP1

1433ECFP0.5/1433EYFP2.5

1433ECFP0.75/1433EYFP2.2

1433ECFP1/1433EYFP2

1433EYFP2

pcDNA3

Detecting 14-3-3 dimerization in Living cells

14-3-3 14-3-3

EYFP

14-3-3

ECFP FRET530nm

430nm

Imaging molecular interactions in single cells using FRET

Confocal laser scanning microscopy

Living cells in medium (no phenol red)With or without stimuli

14-3-3

14-3-3 EYFP

ECFP

Fluorescence microscopy or

Featured with

FRET measured by Inverted Fluorescence Microscopy

•Fluorescence illumination (HBO100 or HBO50) •Fluorescence optics (Plan-Neofluar 10x, 20x, 40x oil, 63x oil, 100x oil) •4 FRET filter cubes (CFP, YFP, FRET, Bleach), highly motorized•Digital camera •Computer •FRET image analysis software •Microscope Setup software

(1) optical fibers

(7,8,9) secondary dichroic

(2) collimators

beam splitters

(3) beam combination

(10) pinhole diaphragm

(4) main dichroic beam

(11) emission filters

splitter

(12) photo-multiplier tubes

(5) scanner mirrors

(13) neutral filters

(6) scanning lens

(14) monitor diode

Laser scanning confocal microscopy

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1

Nature 411, 1065 - 1068 (2001); NAOKI MOCHIZUKI et al.

Ras, small G protein

Activation of RAS by GTP binding

Regulated by guanine nucleotide exchange factor (GEF), the activatorAnd by GTPase activating protein (GAP), the inactivator

Spatial: of the spaceTemporal: of the time

Ras activation near plasma membrane, whereas Rap1 activation near the perinuclear region

Bioluminescence Resonance Energy Transfer BRET

EYFP

Rluc

A

B Substate for Rluc

480 nm

530 nm

Similar to FRET but avoid the photon excitation damage

In jellyfish, blue-light emitting aequorin can promote GFP to excite green light

In the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore

Naturally,

In the current BRET system,

A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins

Proc. Natl. Acad. Sci. USA. 1999 January 5; 96 (1): 151–156By Yao Xu et al.

Whether bacterial circadian proteins form dimers to function?

Biomolecular fluorescence complementation

Origin from classical studies of intragenic complementation of the lacZ locus of E. coli, demonstrating that fragments of -galactosidase that have no enzyme activity can associate spontaneously to generate an active complex

(BiFC)

GFP fragments fused to peptide sequences capable of producing an antiparallel coiled coil produced flurescent complexes in vitro and in E. coli

Biomolecular luminescence/fluorescence complementation

GFPN GFPCGFP

Cut

(1-154) (155-238) Protein A Protein B

A interacts with B? If Yes

Fluorescence complementation

bZIP:basic region-leucine zipper

bZIP family members, such as FOS and JUN

Biomolecular luminescence complementation

Luciferase complementation

N Luc C Luc

A B

N LucC Luc

A B

luciferinlight

A interacts with B

PNAS 101:12288-12293, 2004

FRB: rapamycin-binding domain of the mTOR fused to NLuc

FKBP:FK506-binding protein 12 fused to CLuc

The kinase mTOR is inhibited by FKBP in a rapamycin-dependent manner

From Cell Biology to application in Biomedicine by FRET and BRET

Uncover the molecular interactions in living cells and living animals in a spatial and temporal manner

Molecular diagnosis

Screening drugs in a high throughput way

ReferencesXu, Y. et al. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151-156 (1999)

Mochizuki, N. et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065-1068 (2001)

Jin Zhang et al. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Bio.3, 906-918 (2002).

Hu, C. –D., Chinenov, Y., and Kerppola, T. K. Visualization of interactions among bZIP and Rel family proteins in living cells using biomolecular fluorescence complementation. Mole. Cell 9, 789-798, 2002

Jares-Erijman,E. A., & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387-1395 (2003)

Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Develop. Cell 4, 295-305 (2003)

Kathryn E. Luker et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS 101, 12288-12293 (2004)