objectives for today: why targeted and expressible probes aequorin & gfp mixed with theory
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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. - PowerPoint PPT PresentationTRANSCRIPT
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