PBio/NeuBehav 550: Biophysics of Ca2+ signalingWeek 2 (04/08/13)

Genetically expressible probes and FRET

Objectives for today:• Why targeted and expressible probes• Aequorin & GFP mixed with theory• FRET Theory and photochemistry• The first cameleons• Discuss the 2nd generation cameleon paper

The originalCa/Mg chelator

& buffer

Ca-selective chelator & bufferslow, pH sensitive

Roger Tsien’s fast buffers &fluorescent indicators

Standard tools for calcium studies

KCa ~ 80-300 nM

EDTA (1946)

EGTA (1955)

BAPTA (1980)

Fura, Indo

Ca Green

[–—NP] [Caged calcium][NP-EGTA]

ER

SOC/CRAC channel

SERCA pump

PM Ca2+ ATPase

Na+-Ca2+ exchanger

Plasma membrane

Ca2+

Na+

Ca2+

IP3R channel

Ca2+

Typical Ca2+ fluxes in a non-excitable cell

Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

Ca2+ fluxes in an excitable cell

Inputs: hormones, cytokines, growth factors, antigens

Gq PLC

AgonistR

PIP2

IP3

DAG

ATP

ATP

Ca2+

Ca2+

MitoCa2+

Na+

LDCSG

nucleus

Proteins as indicators

Advantages of proteins as indicators

Highly evolved binding sites

Can be further engineered by mutation

Sophisticated optical properties

Expressed by transfection, infection, transgenic; no loading; do not leak

Targetable to:specific cell types at specific times in organismssubcellular locations and organelles in cells

Genetic targeting of fluorescent constructsTargeting

KDEL

nls

CRsig

Abbreviations:CRsig = calreticulin signal sequenceKDEL = ER retention signaltpA = tissue plaminogen activator (a secreted protein)nls = nuclear localization signalCOX8 = cytochrome oxidase N-terminus

N Cfluorescent protein

Targeted to:

fluorescent protein

fluorescent protein

fluorescent proteinCOX8

fluorescent protein

tpA

cytoplasm

ER

nucleus

mitochondria

secretorygranules

Localization

YC2

YC3er

(Miyawaki et al. & Tsien, Nature, 1997) (Ruzzuto et al. & Tsien, Nature, 1996)

nuGFP and mtBFP

Targeting of fluorescent proteins

scales = "10 m"

---Shimomura O, Johnson FH, Saiga Y, 1962, Extraction, purification and properties of Aequorin, a biolumi-nescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol., 59: 223-239. [470 nm]

---R.Y. Tsien, 1998, The Green Fluorescent Protein, Annual Review of Biochemistry 67, pp 509-544. [508 nm]

Fluorescent proteins make Aequorea glow at 508 nm

Aequorea victoria from Puget Soundin brightfield and false color

Green fluorescent ring

The Nobel Prize in Chemistry 2008. Osamu Shimomura, Martin Chalfie, Roger Y. Tsien

Aequorin 2

Reaction:

Aeq + coelenterazine ----> Aeq.c [non-covalent complex]

Aeq.c + ~3 Ca2+ ----> Ca3.Aeq.c* + CO2

Ca3.Aeq.c* -----> Ca3.Aeq.c** + [blue photon--470 nm]

Aequorin (Aeq) falls in the general heading of "luciferases" that bind a "luciferin" and luminesce in response to a ligand. (The most famous of these is firefly luciferase that can be used to measure ATP concentrations.)

Aequorin is therefore a one-shot calcium detector with a non-linear Ca2+

dependence of luminescence. It is "consumed" by a detection event.

M.W. = 22,514 with four E/F hands

Aequorin: a bioluminescent Ca2+ binding protein complex

containing coelenterazine coelenterazine

ER

SOC/CRAC channel

SERCA pump

PM Ca2+ ATPase

Na+-Ca2+ exchanger

Plasma membrane

Ca2+

Na+

Ca2+

IP3R channel

Ca2+

Stimulating a Ca2+ signal in cytosol & mitochondria

Responses: Fluid secretion, exocytosis, channel gating, enzyme activities, cell division, proliferation, gene expression

Ca2+ fluxes in an excitable cell

Inputs: hormones, cytokines, growth factors, antigens

Gq PLC

Agoniste.g. histamine R

PIP2

IP3

DAG

ATP

ATP

Ca2+

Ca2+

MitoCa2+

Na+

LDCSG

10

5

histamine stimulusre

p ort

ed

[Ca

] (M

)

cytoplasmic Ca is sucked into mitochondria by Δψ Control test:

with 5 M FCCP, Ca does not enter

Aeq targeted insidemitochondrial

matrix

Biological example aequorin

Targeted aequorin reports [Ca] in mitochondrial matrix

HeLa cells transfected with an aequorin construct targeted all the way into the matrix of mitochondria. Cells were then soaked in micromolar coelenterazine at zero calcium for several hours. (Rizzuto...Pozzan, Science, 1998)

protonophore FCCP depolarizes inner membrane of mitochondrion

Δψ

coelenterazine emits 470 nmTyrosine/

phenol: Excit. 275 nm, emits 310 nm)

Why are most proteins not visibly fluorescent?

large box, long wave

small box, short wave

absorptionspectra

"Particle-in-a-box" (think organ pipes)

napthalene anthracene tetracene

UV visible

GFP

GFP: generates a fluorescent chromophore from its amino acids autocatalytically

M.W. = 26,938

dehydration

GFP, a beta barrel

Maturation can be slowEngineer codons folding color photoconversion

N

C

Y66 G67

Engineering color in GFPsExcitation spectra Emission spectra

400 700300 600400 500 500 600

wavelength (nm) wavelength (nm)

Roger Tsien's lab made a range of GFP-derived proteins of different colors by mutation of the expression vector.

Colored GFPs

Flu

ore

sce

nce

inte

nsi

ty

Ab

sorb

ance

4 5 54

Absorption and fluorescence spectra reflect internal energy levels

Absorber has several electronic states (S0, S1, S2, etc.). It also has vibrational states whose close spacing means that photons of a range of close energies can be absorbed. If the absorption spectrum has a second peak (at shorter wavelength), it is for excitation to S2 or because the dye has several molecular forms/conformations.

Absorption bands

S0

S1

En

erg

y

Absorption wavelength

S0

S1

Jablonski diagram

ground state

Green fluorescent protein (GFP) has been engineered to make forms with various fluorescent colors (GFP, CFP, YFP, …). They have overlapping spectra and can transfer excitation directly by FRET when the proteins are close together. The energy transfer occurs without a photon.

Förster/Fluorescence resonance energy transfer (FRET): A proximity detector (molecular ruler) that changes color

FRET illustrate

440 nm

440 nm

480 nm

535 nmFRET!

CFP

CFP

YFP

YFP

Separated:no FRET

Close together:FRET

excitationemission

excitation emission

no 440 nm excitation

hh

h hno h

Forster Eq

FRET depends steeply on distance. R depends on overlap.

440 nm

535 nmFRET!CFP YFP

excitation emissionr

Transfer efficiency E: E = -------------Ro

6

Ro6 + r 6

Förster formula for Förster radius Ro

Ro = Const. {don 2 J n –4} 1/6

Wheredon quantum efficiency of donor orientation factor (0 – 4)n local refractive indexJ "overlap integral" of donor fluorescence (fD) and acceptor absorption A

J =

fD A

500 600

= wavelength

Donor Acceptor

More steps in the Jablonski diagram

absorption(1 fs)

internal conversion

(1 ps)

(polar)solvent

relaxation(100 ps)

competition for re-radiation,quench, FRET,or other non-

radiative (3 ns)

knr hFRET

quenchfluorescence FRET

Donor Acceptor

Fluorescence decays recorded with YC3.1 cameleon dissolved in buffer. Excitation at 420 nm excites the ECFP part. (Habuchi et al. Biophys J, 2002)

FRET speeds donor F and slows acceptor F

480 nm from ECFP

530 nm from EYFP by FRET

time (ns)

em

issi

on in

tens

ity

0 2 4 6

Ca2+-bound CaMeleon

absorption(1 fs)

internal conversion

(1 ps)

(polar) solvent relaxation(100 ps)

competition for re-radiation,quench, FRET

knr hFRET

quenchfluorescenceCFP

FRETYFP

Donor Acceptor

Fluorescence lifetime imaging is a way to image FRET

FRET as a ‘Spectroscopic Ruler’

E % decreases with the distance between donor and acceptor

Förster distance 30 Å

Förster distance 50 Å e.g., ECFP/EYFPFörster distance 70 Å

Two fluorophores separated by Förster distance (r = Ro) have E transfer of 50%

The efficiency of energy transfer is proportional to the inverse of the sixth power of the distance separating the donor and acceptor fluorophore

ECFP/EYFP

x

x

x

x

A family of Ca2+-sensitive switches and buffers

Calmodulin (CaM) : An abundant 149 amino acid, highly conserved cyto-plasmic protein with 4 binding sites for Ca2+ each formed by "EF-hands." Many other homologous Ca2+ binding proteins of this large EF-hand family act as Ca switches and Ca buffers. The Ca2+ ions bind cooperatively and

become encircled by oxygen dipoles and negative charge. CaM com-plexes with many proteins, imparting Ca2+-dependence to their activities.

Calmodulin

KCa ~ 14 M

for free calmodulin

Calmodulin

helix-loop-helix makes

E-F hand{

MW ~ 17 kDa

Calmodulin folds around a target helix

The target peptide in this crystal structure is the regulatory domain of smooth-muscle myosin light-chain kinase (MLCK). The interaction of CaM and MLCK allows smooth muscle contraction to be activated in a Ca2+-dependent manner. (Meador WE, Means AR & Quiocho, 1992.)

MLCK peptide

CaM

4 Ca

Binding of Ca2+ to CaM causes CaM to change conformation. Binding of

CaM to targets can increase the Ca2+ binding affinity of CaM greatly.

Calmodulin folds

Two GFPs in one peptide interact by fluorescence resonance energy transfer (FRET). Targeting sequences can be added to direct constructs to specific compartments. (Miyawaki, Roger Tsien et al., 1997)

Design of CaMeleons:Expressible proteins for Ca detection

Design of CaMeleons:

440 nm

FRET

CFP

CFP

YFP

YFP

Low calcium:No FRET

High calcium:FRET

CaMMLCK

NC

N

C

480 nm

535 nm

440 nm

Cano Ca

YC3.1cameleon

emis

sion

inte

nsity

Note two peaks

Ca-sensitive cameleon emission spectra

Emission wavelength (nm)

moreFRET

(Miyawaki, Roger Tsien et al., 1997)

Cameleon emission combines two spectra

ECFPEYFP

emission

ECFPEYFP

There is FRET even with no Ca2+! Amount of FRET gives distance changes. It is not a large change.

Cano Ca

YC3.1cameleon

emis

sion

inte

nsity

Ca-sensitive FRET reporter. How do calciums bind?

Calcium binding and the conformation change can be tailored by making mutations in the EF hand regions of the calmodulin. Glutamate E31 is in the first EF hand (at p12') and E104 is in the third EF hand (also at p12').

GC1

GC1/E104Q

GC1/E31Q

510

/445

nm

em

issi

on

ra

tio

1.0

green cameleon 1 fluorescence ratios

free calcium (M)

NC

E31

E104

(Miyawaki et al., 1997)

higher affinitylower affinity

ER-directed Cameleon

PC12 cells are transfected with D1-ER, a Roger Tsien cameleon directed to the ER. SERCA pump blocker BHQ shows efflux, ATP shows efflux with a transient refilling by outside Ca due to SOCE. ATP makes IP3 production,

(Dickson,....,Hille, 2012)

Miyawaki et al. 1999 paperDynamic and quantitative Ca2+ measurements using

improved cameleons

Each figure will be described by a student--as if you are teaching it to us for the first time.

Further questions will come from the audience.

--5 min per fig--one panel at a time--give it a title--explain axes and subject--ask leading questions to get students to discuss--what is being tested and what is concluded?

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Fig 1

0.1

0.0

2.12

2.1

2Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Y66 G67

2.13.1

YC2.1

2.1 3.1

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Emission wavelength (nm)

Fig 2AB

Fig 2CD

2.13.1

2.1

3.1

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Fig 3

YC2.1YC2

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Fig 4 YC2.1

YC3.1

500 uM

150 uM

40 uM

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin

Fig 5

YC3.1+- CaM

CaM

split

2.1

2.1

3.1

split 2.1

Emission wavelength (nm)

Fig 1. Andrea McQuateFig 2a,b. Jacob BaudinFig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse MacadangdangFig 5. Jerome Cattin