PBio/NeuBehav 550: Biophysics of Ca2+ signalingWeek 2 (04/04/11)

Genetically expressible probes and FRET

Objectives for today:• Motivation for targeted and expressible probes• Aequorin & GFP• FRET Theory and photochemistry• The first cameleons• Discuss recent Calsequestrin 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]

Proteins as indicators

Advantages of proteins as indicators

Binding site already evolved for high selectivity

Can be further engineered by directed or random mutation

Genetically expressibleby transfection, viral infection, transgenic lines

Targetable to specific cell types at specific times in organismsuse promoters and enhancers

Targetable to subcellular locations and organelles in cellsadd nuclear, mitochondrial, ER, etc. targeting sequences

No need for pipettes or tricky loading procedures

Do not leak out of cell or organelle

Some are more resistant to bleach than conventional dyes

---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

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+

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

Biological example aequorin

Aequorin reports [Ca] outside and inside mitochondria

Two batches of HeLa cells transfected with different aequorin constructs. One targets to the inter-membrane space of mitochondria and the other targets all the way to the matrix of the mitochondria. Cells were then soaked in micromolar coelenterazine at zero calcium for several hours. (Rizzuto...Pozzan, Science, 1998)

0

4

10

5histamine stimulus

histamine stimulus

time

[Ca

] (M

)

[Ca

] (M

)

+ 5 M FCCP: Ca does not enter

Control: Ca sucked into mitochondrion

Aeq targeted insidemitochondrial matrixAeq targeted between

mitochondrial membranes

+ 5 M FCCP

Mito

FCCP will depolarize inner membrane of mitochondrion

Control

coelenterazine emits 470 nm

Spectra and particle-in-a-box (think organ pipes)

napthalene anthracene tetracene

large box, long wave

small box, short wave

absorptionspectra

Tyrosine/phenol: Exc. 275 nm, emits 310 nm)

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

Note: N- and C- are close to each other

Ph sensitive quantum yield of FPs

Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM. mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE. 2008;3:e3944.

pKa = 6.6

Dark form

Photoswitchable FPs

Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM. mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE. 2008;3:e3944.

pKa = 6.6

405 nmAstrocytes mKikGR-actin

after 405 nm

Dark form

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

FRET as a ‘Spectroscopic Ruler’

E % decreases as distance between

donor and acceptor increases

Förster distance 30 Å

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

When two fluorophores separated by Förster distance (where r = Ro), E transfer is 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

How many Ca need to bind?

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

Genetic tailoring of first-generation cameleonsTargeting

KDEL

KDEL

KDEL

nls

CRsig

CRsig

CRsig

CaM-M13

EGFP

EGFP

EGFP

EYFP

EYFP

EYFP

Cameleon name:

2

2nu

3er

YC2

YC3er

YC4er

Abbreviations:CRsig = calreticulin signal sequencenls = nuclear localization signalKDEL = ER retention signal (Miyawaki et al. and Tsien, Nature, 1997)

N C

Localization

Targeting of cameleons

YC2

YC3erGC2nu

scales = "10 m"

How many calciums bind? Mutating calcium binding.

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

% o

f 51

0/4

45

nm

em

issi

on r

atio

100green cameleon 1 fluorescence ratios

free calcium (M)

N C

YC2.1 YC4.3E31QD1YC3.3

E104Q

Mutating calcium binding for ER/SR sensitivity

Palmer AE, Jin C, Reed JC, Tsien RY. PNAS 2004

ER/SR

Canato et al. 2010 paperMassive alterations of sarcoplasmic reticulum free

calcium in skeletal muscle fibers lacking calsequestrinrevealed by a genetically encoded probe

Sarah Nowakowski: Explain Question that paper investigates (not what it concludes). Explain SR and CSQ. No need for audience inclusion.

Curtis Easton: Fig. 1A&BMerle Gilbert: Fig. 1CAaron Williams: Fig. 2Wucheng Tao: Fig. 3Jennifer Deem: Fig. 4 Bertil: Fig. 5

--5 min per fig--give it a title--explain axes--ask leading questions to get students to discuss

Sarah Nowakowski: Explain Question that paper investigates (not what it concludes). Explain SR and CSQ. No need for audience inclusion.

Massive alterations of SR free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe

CASQ2 gene expression in Allen Brain Atlas

Cerebellar Pukinje neurons

(mouse CASQ2 ISH)

(mouseatlas)

(A) Localization of D1ER on both sides of Z lines stained with α-actinin antibody (D1ER, green; α-actinin antibody + rhodamine, red).

Canato M et al. PNAS 2010;107:22326-22331

D1ER -actinin

Fig 1 A&B; Curtis Easton

(A) Localization of D1ER on both sides of Z lines stained with α-actinin antibody (D1ER, green; α-actinin

antibody + rhodamine, red).

Canato M et al. PNAS 2010;107:22326-22331

D1ER

Fig 1 C; Merle Gilbert

Typical recordings of changes in intraluminal SR Ca2+ concentrations during repetitive stimulation at different stimulation rates.

Canato M et al. PNAS 2010;107:22326-22331

WT CSQ-KO

Fig 2 A, B, C; Aaron Williams

YFP

CFP1 Hz

5 Hz

20 Hz

Average amplitudes of the decline in the YFP/CFP R (ΔR) during contractile activity with increasing stimulation frequency.

Canato M et al. PNAS 2010;107:22326-22331

Fig 3; Wucheng Tao

Time course of the change in the YFP/CFP R during the SR refilling.

Canato M et al. PNAS 2010;107:22326-22331

YFP

YFP

YFP/CFP

YFP/CFP

both areCSQ-KO

fibers

Fig 4; Jennifer Deem

Fibers lacking CSQ are not able to maintain a high cytosolic calcium concentration during repetitive stimulations.

Canato M et al. PNAS 2010;107:22326-22331

WT CSQ-DKO

20-Hz stimulation

Fu

ra-2

Fu

ra-2

Fig 5; Bertil

Cameleon emission combines two spectra

ECFPEYFP

emission

ECFPEYFP

There is FRET even with no Ca2+! Amount of FRET gives distances: ~5.0 and 6.5 nm, or 50 and 65 Å. This is not a large change.

Cano Ca

YC3.1cameleon

emis

sion

inte

nsity

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

480 nm from ECFP

530 nm from EYFP by FRET

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

time (ns)

em

issi

on in

tens

ity

FRET speeds donor and slows acceptor F

0 2 4 6

Ca2+-bound CaMeleon

R2' R7'

R5

R6

Electron withdrawingon Fluo dye series