PBio/NeuBehav 550: Biophysics of Ca
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PBio/NeuBehav 550: Biophysics of Ca 2+ signaling Week 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.

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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


Tools for calcium studies

Standard tools for calcium studies

Tools for calcium studies

[Caged calcium]

[NP-EGTA]

[–—NP]

The original

Ca/Mg chelator

& buffer

EDTA (1946)

EGTA (1955)

BAPTA (1980)

Fura, Indo

Ca Green

Ca-selective

chelator & buffer

slow, pH sensitive

Roger Tsien’s

fast buffers &

fluorescent

indicators

KCa ~ 80-300 nM


Ca 2 fluxes in an excitable cell
Ca2+ fluxes in an excitable cell

Typical Ca2+ fluxes in a non-excitable cell

Inputs: hormones, cytokines, growth factors, antigens

PIP2

Agonist

Na+-Ca2+ exchanger

R

DAG

Gq

PLC

IP3

LDCSG

Ca2+

Ca2+

Ca2+

IP3R channel

SERCA

pump

Na+

ATP

nucleus

ER

Plasma membrane

Ca2+

ATP

Ca2+

Mito

PM Ca2+ ATPase

SOC/CRAC channel

Ca2+

Na+

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


Proteins as indicators
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 organisms

subcellular locations and organelles in cells


Targeting

Genetic targeting of fluorescent constructs

Targeting

Targeted to:

cytoplasm

N

C

fluorescent protein

ER

KDEL

CRsig

fluorescent protein

secretory

granules

fluorescent protein

tpA

nucleus

nls

fluorescent protein

mitochondria

COX8

fluorescent protein

Abbreviations:

CRsig = calreticulin signal sequence

KDEL = ER retention signal

tpA = tissue plaminogen activator (a secreted protein)

nls = nuclear localization signal

COX8 = cytochrome oxidase N-terminus


Localization
Localization

Targeting of fluorescent proteins

scales = "10 mm"

YC2

nuGFP and mtBFP

YC3er

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

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


Aequoria
Aequoria

Fluorescent proteins make Aequorea glow at 508 nm

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

Green fluorescent ring

---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 FluorescentProtein, Annual Review of Biochemistry 67, pp 509-544. [508 nm]

Aequorea victoria from Puget Sound

in brightfield and false color


Aequorin 2
Aequorin 2

Aequorin: a bioluminescent

Ca2+ binding protein complex containing coelenterazine

coelenterazine

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

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.)

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

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


Ca 2 fluxes in an excitable cell1
Ca2+ fluxes in an excitable cell

Stimulating a Ca2+ signal in cytosol & mitochondria

Inputs: hormones, cytokines, growth factors, antigens

PIP2

Agonist

e.g. histamine

Na+-Ca2+ exchanger

R

DAG

Gq

PLC

IP3

LDCSG

Ca2+

Ca2+

Ca2+

IP3R channel

Na+

SERCA pump

ATP

ER

Plasma membrane

Ca2+

ATP

Ca2+

Mito

PM Ca2+ ATPase

SOC/CRAC channel

Ca2+

Na+

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


Biological example aequorin
Biological example aequorin

Targeted aequorin reports [Ca] in mitochondrial matrix

protonophore FCCP depolarizes inner membrane of mitochondrion

Aeq targeted inside

mitochondrial matrix

Δψ

histamine stimulus

10

cytoplasmic Ca

is sucked into

mitochondria

by Δψ

reported [Ca] (mM)

5

Control test:

with 5 mM FCCP,

Ca does not enter

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)


Why are most proteins

not visibly fluorescent?

coelenterazine

emits 470 nm

Tyrosine/

phenol:

Excit. 275 nm,

emits 310 nm)

napthalene anthracene tetracene

"Particle-in-a-box"

(think organ pipes)

absorption

spectra

UV visible

small box, short wave

large box, long wave


GFP

GFP: generates a fluorescent chromophore

from its amino acids autocatalytically

Y66 G67

Maturation can be slow

Engineer codons

folding

color

photoconversion

M.W. = 26,938

N

dehydration

C

GFP, a beta barrel


Colored gfps
Colored GFPs

Engineering color in GFPs

Excitation spectra

Emission spectra

4

5

5

4

Absorbance

Fluorescence intensity

300

400

500

600

400

500

600

700

wavelength (nm)

wavelength (nm)

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


Absorption bands

Absorption and fluorescence spectra reflect internal energy levels

Absorption bands

S1

S1

Energy

S0

S0

ground state

Jablonski diagram

Absorption

wavelength

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.


Fret illustrate

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

FRET illustrate

440 nm

480 nm

YFP

hn

CFP

emission

hn

Separated:

no FRET

excitation

no 440 nm excitation

no hn

440 nm

hn

hn

YFP

FRET!

CFP

535 nm

Close together:

FRET

excitation

emission

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.


Forster eq
Forster Eq levels

FRET depends steeply on distance. R depends on overlap.

Donor Acceptor

440 nm

YFP

FRET!

CFP

535 nm

excitation

emission

r

fDeA

Ro6

Ro6 + r 6

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

Förster formula for Förster radius Ro

Ro = Const. {fdonk2J n –4} 1/6

Where

fdon quantum efficiency of donor

k orientation factor (0 – 4)

n local refractive index

J "overlap integral" of donor fluorescence (fD) and acceptor absorption eA

J =

500 600

l = wavelength


More steps in the Jablonski diagram levels

internal

conversion

(1 ps)

(polar)

solvent

relaxation

(100 ps)

competition

for re-radiation,

quench, FRET,

or other non-

radiative (3 ns)

absorption

(1 fs)

knr

hnFRET

fluorescence

quench

FRET

Donor

Acceptor


Lifetime fret
Lifetime & FRET levels

FRET speeds donor F and slows acceptor F

Ca2+-bound CaMeleon

competition

for re-radiation,

quench, FRET

(polar) solvent

relaxation

(100 ps)

530 nm from

EYFP by FRET

emission intensity

internal

conversion

(1 ps)

absorption

(1 fs)

knr

480 nm from

ECFP

hnFRET

Donor

Acceptor

Fluorescence lifetime imaging is a way to image FRET

quench

fluorescence

CFP

FRET

YFP

0 2 4 6

time (ns)

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 as a spectroscopic ruler
FRET as a ‘Spectroscopic Ruler’ levels

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

Förster distance 30 Å

Förster distance 50 Å e.g., ECFP/EYFP

Förster distance 70 Å

E % decreases with the distance between donor and acceptor

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


Calmodulin

A family of Ca levels2+-sensitive switches and buffers

Calmodulin

helix-loop-helix makes E-F hand

{

x

x

x

x

KCa ~ 14 mM

for free calmodulin

Calmodulin

MW ~ 17 kDa

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 folds

Calmodulin folds around a target helix levels

Calmodulin folds

MLCK

peptide

4 Ca

CaM

Binding of Ca2+ to CaM causes CaM to change conformation. Binding of CaM to targets can increase the Ca2+ binding affinity of CaM greatly.

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.)


Design of cameleons

Design of CaMeleons: levels

Expressible proteins for Ca detection

Design of CaMeleons:

440 nm

480 nm

YFP

Low calcium:

No FRET

C

N

CaM

MLCK

CFP

C

YFP

440 nm

FRET

CFP

High calcium:

FRET

535 nm

N

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)


Ca sensitive cameleon emission spectra
Ca-sensitive cameleon emission spectra levels

Note two peaks

no Ca

Ca

YC3.1

cameleon

emission intensity

more

FRET

Emission wavelength (nm)

(Miyawaki, Roger Tsien et al., 1997)


Cameleon emission combines two spectra
Cameleon emission combines two spectra levels

EYFP

ECFP

Ca

no Ca

YC3.1

cameleon

emission intensity

emission

ECFP

EYFP

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


Ca sensitive fret reporter how do calciums bind
Ca-sensitive FRET reporter. How do calciums bind? levels

(Miyawaki et al., 1997)

green cameleon 1 fluorescence ratios

1.0

E104

C

N

E31

lower

affinity

higher affinity

GC1

510/445 nm emission ratio

GC1/E31Q

GC1/E104Q

free calcium (M)

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').


ER-directed levelsCameleon

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

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,


Miyawaki et al. 1999 paper levelsDynamic and quantitative Ca2+ measurements using improved cameleonsEach 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 Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin


Fig 1 levels

G67

Y66

0.1

0.0

2.1

2

2.1

Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin

2


Fig 2AB levels

YC2.1

2.1

3.1

Emission wavelength (nm)

3.1

2.1

Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin


Fig 2CD levels

3.1

Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin

2.1

2.1

3.1


Fig 3 levels

YC2.1

YC2

Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin


Fig 4 levels

YC2.1

500 uM

150 uM

YC3.1

40 uM

Fig 1. Andrea McQuateFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin


Fig 1. Andrea McQuate levelsFig 2a,b. Jacob Baudin

Fig 2c,d. Anastasiia StratiievskaFig 3. Benjamin DrumFig 4. Jesse Macadangdang

Fig 5. Jerome Cattin

Fig 5

CaM

split 2.1

2.1

3.1

split 2.1

YC3.1

+- CaM

Emission wavelength (nm)


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