biomedsci 231 27 february 2008 fluorescence in the study of membrane proteins
DESCRIPTION
BioMedSci 231 27 February 2008 Fluorescence in the study of membrane proteins Counting transporter molecules (sildes 2-18) FRET analysis of nicotinic receptor assembly (19-32 Fluorescence imaging ratio of nicotinic receptor assembly (33-34) A flourescent unnatural amino acid (35-39). - PowerPoint PPT PresentationTRANSCRIPT
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BioMedSci 231 27 February 2008
Fluorescence in the study of membrane proteins
1. Counting transporter molecules (sildes 2-18)2. FRET analysis of nicotinic receptor assembly (19-32
3. Fluorescence imaging ratio of nicotinic receptor assembly (33-34)4. A flourescent unnatural amino acid (35-39)
Henry Lester
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Biological Steps Calibration steps
(1) Design and construct several GFP-containing constructs of the transporter protein.
(2) Test the construct for normal function.
(3) Test the construct, as well as possible, for normal localization and targeting.
(4) Obtain a genomic clone containing the relevant portion of the protein.
(5) Construct an exon replacement targeting vector containing the GFP fusion construct.
(6) Generate embryonic stem cells harboring the gene for the GFP-protein in place of the wild type protein.
(7) Construct knock-in mice.
(8) Study the wild-type, heterozygote, and homozygote mice qualitatively and quantitatively to confirm comparable localization and expression levels.
(1) Express milligram quantities of the same GFP only (unlinked to the membrane protein).
(2) Study the GFP in single-molecule fluorescence.
(3) Couple the GFP to agarose beads.
(4) Define the GFP density on the beads in terms of (2).
Measurement Steps
(1) Study appropriate preparations from the knock-in mice, using tissue slices, and dissociated cells.
(2) Calculate the protein-GFP density by standardizing against the beads.
Strategy for quantitative data on transporter localization at synapses
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COOH
NH2
A fusion protein: mouse GABA transporter (mGAT1)-GFP
4/39
A C-terminal mGAT1-GFP fusion localizes partially to the membrane when expressed in HEK 293 cells
GFP
NH2
CO2-
mGAT1-GFP
CO2-
GFP-mGAT1
NH2
5/39
mGAT1-GFP has functional characteristics identical to mGAT1when tested in HEK 293 cells
6/39
Intron 14Exon 15
part of mGAT1 genomic DNA
mGAT1-GFP fusion construct for homologous recombination
pKO 907 Diphtheria toxin
neo
loxP loxP
spacer-GFP-stop
DT
stop
7/39
Exon 15
part of mGAT1 genomic DNA
mGAT1-GFP fusion construct for homologous recombination
4.5 kb
1 2 3 M 4 5 6
PCR screening identifies ES cells carrying the mutant gene.
Intron 14
pKO 907 Diphtheria toxin
neo
loxP loxP
DT
spacer-GFP-stop
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Fluorescence in the brain of an mGAT1-GFP knock-in mouse
cerebellum
9/39
Molecular layer (basket cells stain)
Purkinje cell layer“pinceux” stain heavily
Granule cell layer
<Immunocytochemistry(Radian et al)
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Molecular layer (basket cells stain)
Purkinje cell layer“pinceaux” stain heavily
Granule cell layer
<Immunocytochemistry(Radian et al)
GFP fluorescence >
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P
P
PGranule cell layer
Molecular layer
Neo-deleted het, 29-days-old, Cerebellum
50.0 m
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GAT1-GFP expression in cerebellum: basket cell terminals in molecular layer
50 m
13/39
B
C
A
D
-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.60
20
40
60
80
100
120
Re
lati
ve
flu
ore
sc
en
t in
ten
sit
y (
%)
D istance (m)
untreated boutonstranslocation treatmentbeads
0.5 M
The data suggest:
the intracellular GAT1-GFP
(31-33% of GAT1 in WT; 80%
in mGAT1-GFP)
is so close to the membrane
that it appears membrane-
bound in the confocal
microscope.
14/39
Schematic drawing of a chandelier cell based on immunostaining for parvalbumin
in human neocortex
Ch terminals
from Felipe et al, Brain (1999) 122, 1807
15/39
Calibrations: Transparent 90-m dia beads with calibrated surface densities of EGFP
Incubate Ni-NTA beads with measured
numbers of His6 EGFP molecules
Include beads with EGFP-labeled cells in the fluorescent microscope
various[His6GFP]
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Single-molecule fluorescence at increasing GFP densities
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Single-molecule and macroscopic measurements of GFP density
- absolute calibration - accurate within 20% - over 4 orders of magnitude - useful on both wide-field and confocal microscopes
0 50 100 150 200 2500
5
10
15
20
25
30
35
40
Fre
quen
cy
Intensity (counts/GFP•s)
100 101 102 103 104 105102
103
104
105
106
107*
*
Macroscopic measurements Slope = 1
Inte
nsity
(co
unts
/m
2 *s)
Expected density (GFP/m2)
10-11 10-10 10-9 10-8 10-7
[His6-GFP], M
100 101 102 103 1040.00.20.40.60.81.01.21.41.61.8
Mea
sure
d/ex
pe
cte
d in
tens
ity
Expected density (GFP/m2)
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Molecular layer of cerebellum
Total GAT1/bouton: 9000Volume density 5000 GAT1/m3, Surface density 1340 GAT1/m2.
Cartridge in cortexcontains 365,000 GAT1
Pinceaux in cerebellum 7.8 million GAT1,at a volume density of 7700 GFP/m3
Axons: 640 GAT1/m
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Binding region
Membrane region
Cytosolicregion
(incomplete)
Colored by secondary
structure
Colored by subunit(chain)
Nearly Complete Nicotinic Acetylcholine Receptor, a Well-Studied Cys-loop Receptor
~ 2200 amino acids in 5 chains
(“subunits”),
MW ~ 2.5 x 106
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+ +
Fre
e E
nerg
y
Reaction Coordinate
Free subunits
Increasingly stable
assembled states
Nicotine may stabilize subunit interfaces
15 M Nicotine+
1 M Nicotine+
(pKa = 7.9)
0 mV-70 mV
Nicotine accumulates in cells
+Boundstates with
increasing affinity
Fre
e E
nerg
y
Reaction Coordinate
C
AC
A2C A2O
A2D
Highest affinity bound state
unbound
Binding eventually favors high-affinity states
Upregulation as a thermodynamic consequence of nicotine-receptor Interactions
Nicotine
hr0 20 40 60
Incr
ease
d H
igh-
Sensi
tivit
y R
ece
pto
rs
RLS RHS
Covalently stabilized
AR*HSDegradation
+ nicotine
?
Fluorescence assays for receptor changes in response to chronic nicotine?
21/39
4-YFP-N1
4-YFP-N2
4-YFP-M
HA tag
XmaI (34 aa)
YFP
HA* tag
XmaI (34 aa)
YFPHA tag
HA tag
BStEII (426 aa)
YFP
M2M1 M3 M4Ligand-binding domain IC loop
1 aa 629 aa4 nAChR
c-myc tag CFP
2-CFP-C
c-myc tag
PPUMI (381 aa)
CFP 2-CFP-M
1 aa 501 aa 2 nAChR
Candidate fluorescently tagged 4 and 2 subunit constructs
YFP
CFP
Endocytosis motifs: YXXM, L, F; LL ER retention motifs: R/K-X-R/K, R/K-R/KAmphipathic helix contains ER export motifs: Ds and Es
PKAPKCTyrosine kinaseCasein kinaseCalmodulin dependent kinase IICyclin dependent kinase 5
2 M3-M4 IC Loop
CFP400
440
459
360
4 M3-M4 IC Loop
YFP
520
560
600
602
400
440
480
360 VHHRSPTTHT MAPWVKVVFL EKLPTLLFLQ QPRHRCARQR
LRLRRRQRER EGAGTLFFRK GPAADPCTCF VNPASMQGLA
GAFQAEPAAA GLGRSMGPCS CGLREAVDGV RFIADHMRSE
DDDQSVREDW KYVAMVIDR
VHHRSPRTH TMPAWVRRVF LDIVPRLLFM
KRPSVVKDNC RRLIESMHKM ANAPRFWPEP ESEPGILGDI
CNQGLSPAPT FCNRMDTAVE TQPTCRSPSH KVPDLKTSEV
EKASPCPSPG SCHPPNSSGA PVLIKARSLS VQHVPSSQEA
AEGSIRCRSR SIQYCVSQDG AASLTESKPT GSPASLKTRP
SQLPVSDQTS PCKCTCKEPS PVSPITVLKA GGTKAPPQHL
PLSPALTRAV EGVQYIADHL KAEDTDFSVK EDWKYVAMVI
DR
We avoided targeting and signaling motifs
VHHRSPTTHT MAPWVKVVFL EKLPTLLFLQ QPRHRCARQR
LRLRRRQRER EGAGTLFFRK GPAADPCTCF VNPASMQGLA
GAFQAEPAAA GLGRSMGPCS CGLREAVDGV RFIADHMRSE
DDDQSVREDW KYVAMVIDR
400
440
459
360 VHHRSPRTH TMPAWVRRVF LDIVPRLLFM
KRPSVVKDNC RRLIESMHKM ANAPRFWPEP ESEPGILGDI
CNQGLSPAPT FCNRMDTAVE TQPTCRSPSH KVPDLKTSEV
EKASPCPSPG SCHPPNSSGA PVLIKARSLS VQHVPSSQEA
AEGSIRCRSR SIQYCVSQDG AASLTESKPT GSPASLKTRP
SQLPVSDQTS PCKCTCKEPS PVSPITVLKA GGTKAPPQHL
PLSPALTRAV EGVQYIADHL KAEDTDFSVK EDWKYVAMVI
DR
520
560
600
602
400
440
480
360
Ubiquitination motifs: K
Raad Nashmi
22/39
ECFPmECFP
XFP =EYFP
mEYFPVenus
mVenusCerulean
mCeruleanEGFP
mEGFPmCherry
λ
Ligand binding M1 M2 M3 M4M3-M4 loop
aa 1 aa 629
M4
M3 - M4 loop
α4 β2aa 1 aa 501
c-myc tag XFP
α4-XFP-M β2-XFP-M
HA tag XFP
A collection of fluorescent Cys-loop receptor subunits
FRET pairs
α6 β3
Analogous functional but less extensive fluorescent series
C. elegans GluClα, Gluclβ
(dopaminergic neurons) (selective neuronal silencing
m = monomeric
23/39
Raad Nashmi
YFP,
Leu9-Ala-YFP,
CFP
There are 3C2 = 6 interesting genotypes of
fluorescent nAChR 4 mutants
All phenotypes are viable &
neo-deleted
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A. Decrease in donor fluorescence or quantum yield: E = (FD - FDA)/FD
1. Need FD, donor fluorescence under identical conditions but with no acceptor nearby, i.e. before acceptor added or after acceptor bleached or split off
B. Decrease in donor excited state lifetime: E = (D - DA)/D
C. Decrease in rate of donor photobleaching
D. Sensitized emission from the acceptor is proportional to EQA
1. Sensitized emission generally contaminated by long-wavelength tail of donor emission and direct excitation of acceptor; both must be deducted by multiwavelength measurements
2. For r << R0, sensitized emission may disappear due to other quenching mechanisms operating at very short distances
E. FRET-mediated emission from the acceptor is less polarized than if acceptor had been directly excited – useful for detecting FRET between identical molecules (homotransfer, donor-donor transfer. “anti-Stokes” effect)
F. Distinguish FRET from trivial absorption followed by re-emission
From Roger Tsien’s notesII. How is FRET detected and measured?
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Lakowicz 13-13
Older microscopes could image at only a few wavelengths, using interference filters
26/39
Lakowicz 13-13
Modern microscopes can record an entire spectrum (at 5 nm intervals) for each pixel,Enabling us to analyze the relative amount of each fluorescent component
Spectrum = a(donor) + b(acceptor)
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FRET is useful because R0 is on the order of protein sizes (16 -56 Ǻ)
Efficiency of energy transfer E is the fraction of photons absorbed by the
donor which are transferred to the acceptor.
1. Therefore a nonfluorescent donor can still participate in RET
2. Poorer RET smaller R0
Lakowicz 13-1, 2
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FRET measurements by acceptor photobleaching (donor dequenching)
0 20 40 60 YFP intensity decrease (%)
Intercept = 0.24
FRET efficiency = 1- (1/(1 + Intercept)) = 19.4%
80 100
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Fluorescent subunits tell us about nicotinic receptor assembly
4Y/
4C/2
4/2
Y/2C
6Y/
6C/2
4/2
/3Y/3
C
*
Ryan DrenanNeuro2a
30/39
Theory of FRET in pentameric receptors with αnβ(5-n) subunits
0
20
40
60
80
0 20 40 60 80 100Distance between adjacent subunits, A
FR
ET
Eff
icie
ncy 100%(4)3(2)2
100%(4)2(2)3
y = -0.1685x + 18.96
y = 0.1685x + 2.11
0
4
8
12
16
20
0 20 40 60 80 100percent of 2-subunit receptors
FR
ET
eff
icie
nc
y100% α3β2100% α2β3
% receptors with α3
No FRET
No FRETE
1/2 1/4 1/4
E1 E2 E3 E4
1/8
1/4
1/4
1/8 1/8 1/8
50% α-CFP, 50% α-YFP
b/a =1.62; 1.62-6 = 0.055
31/39Cagdas Son
Data:Changes in subunit stoichiometry caused by varying subunit expression levels
0
2
4
6
8
10
12
14
16
18
ratio,(4CFP + 4YFP) : 21:91:41:1
% F
RE
T E
ffic
ien
cy
4:1
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The key experiment: changes in subunit stoichiometry caused by chronic nicotine!
Cagdas Son
0
2
4
6
8
10
12
14
16
(4CFP + 4YFP) : 21:1
+ Nicotine
% F
RE
T E
ffic
ienc
y
control
4(2CFP + 2YFP)1:1
+ Nicotinecontrol
33/39
When x X1FP-tagged molecules and y X2FP molecules are co-expressed in a cell, the intensities of X1FP and X2FP can be calculated as:
Fe(X1FP) = xC1 ; Fe(X2FP) = yC2 where Fe(X1FP) and Fe(X1FP) are (X1FP) and (X2FP) intensities, calculated by acceptor photobleaching. Here, X1FP is the donor. The Fe(X1FP) corresponds to the dequenched X1FP intensity when 100% of the acceptor molecules are bleached. Thus this value represents X1FP carrying subunits participating in assembled pentamers with X2FP containing subunits. Similarly the X2FP intensity detected by exciting X1FP (for CFP, at 439) nm and detecting the X2FP emission due to FRET, hence arises from X2FP containing subunits participating in assembled pentamers with X1FP containing subunits. Both intensities are detected by spectral imaging and unmixed to eliminate background fluorescence and the overlap of emission spectra. C1 and C2 are constants reflecting the laser intensities, the system transfer function, the properties of the fluorophores, etc. The fluorescence intensity ratio is defined as k1 = Fe(X1FP) / Fe(X2FP) = C x/ y where C is a constant that equals to C1 / C2 . Similarly, coexpressing x X2FP and y X1FP subunits yields a fluorescence intensity ratio k2 = Fe(X1FP) / Fe(X2FP) = C y / x From these two equations, both the subunit ratio and the constant C can be determined: x / y = k1 / k2; C = k1 k2
The method requires a complete set: 4X1FP, a4 X2FP at equal densities; 2X1FP, 2X2FP.
Fluorescence Intensity Ratio Analysis of Subunit Stoichiometry(Hummer, Delzeith, Gomez, Moreno, Mark, & Herlitze (2003), JBC 278, 49386
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cDNA:1
Fluorescence Intensity Ratios Also Yield Overall Subunit Stoichiometry: 42= √(k1/k2)
CF
P i
nte
nsi
ty
2YFP4CFP
[Nic]
Mtotal 24
%(4)2(2)3
0 1.08 70
1 1.45 95
Cagdas SonYFP intensity
2CFP4YFP
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The in vivo Nonsense Suppression Method for Unnatural Amino Acid Incorporation
Electroporate into cultured
mammalian cells
Inject intoXenopus oocyte
OR
Functional measurements on
the protein containing the
unnatural residue
Im Vm
Im / Vm
Ribosome incorporates unnatural
amino acid; protein reaches
membrane (12-48 h)
mix
STOP (“nonsense”) codon(UAG = “amber”)
mRNA or cDNA
1Gene for the protein with “stop” codon at
site of interest
2“amber suppressing”
tRNA based on Tetrahymena thermophila
b. Appropriate anticodon
a. Unnatural amino acid chemically appended
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Lys-BODIPYFL: a fluorescent unnatural amino acid
4-base codon method
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Lys-BODIPYFL: a fluorescent unnatural amino acid
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Counting photobleach steps to assess stoichiometry