Transcript Lester-UCSDBiomedSci 231-Mar

BioMedSci 231 27 February 2008
Fluorescence in the study of membrane proteins
3.
1. Counting transporter molecules (sildes 2-18)
2. FRET analysis of nicotinic receptor assembly (19-32
Fluorescence imaging ratio of nicotinic receptor assembly (33-34)
4. A flourescent unnatural amino acid (35-39)
Henry Lester
1/39
Strategy for quantitative data on transporter localization at synapses
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.
(1) Express milligram
quantities of the same GFP
only (unlinked to the
membrane protein).
(2) Study the GFP in singlemolecule fluorescence.
(3) Couple the GFP to agarose
beads.
(4) Define the GFP density on
the beads in terms of (2).
(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.
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.
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A fusion protein:
mouse GABA transporter (mGAT1)-GFP
NH2
COOH
3/39
A C-terminal mGAT1-GFP fusion localizes partially to the membrane
when expressed in HEK 293 cells
NH2
CO2CO2NH2
GFP-mGAT1
GFP
mGAT1-GFP
4/39
mGAT1-GFP has functional characteristics identical to mGAT1
when tested in HEK 293 cells
2.5 M GABA
4
GABA uptake (pmol/g protein/10 min)
GABA uptake (pmol/g protein)
2.0
1.5
mGAT1-GFP
mGAT1
GFP
1.0
0.5
0.0
3
mGAT1-GFP
mGAT1
GFP
Non-transfected
2
1
0
0
10
20
30
40
Time (min)
50
60
0
20
40
60
80
100
120
[GABA] M
5/39
mGAT1-GFP fusion construct for homologous recombination
part of mGAT1 genomic DNA
Exon 15
Intron 14
spacer-GFP-stop
DT
neo
pKO 907
stop
Diphtheria toxin
loxP
loxP
6/39
mGAT1-GFP fusion construct for homologous recombination
part of mGAT1 genomic DNA
Exon 15
Intron 14
spacer-GFP-stop
neo
pKO 907
loxP
loxP
DT
Diphtheria toxin
1 2 3 M 4 5 6
4.5 kb
PCR screening identifies ES
cells carrying the mutant gene.
7/39
Fluorescence in the brain of an mGAT1-GFP knock-in mouse
cerebellum
8/39
<Immunocytochemistry
(Radian et al)
Molecular layer (basket cells stain)
Purkinje cell layer
“pinceux” stain heavily
Granule cell layer
9/39
<Immunocytochemistry
(Radian et al)
GFP fluorescence >
Molecular layer
(basket cells stain)
Purkinje cell layer
“pinceaux” stain heavily
Granule cell layer
10/39
Neo-deleted het, 29-days-old, Cerebellum
P
Granule cell layer
P
P
Molecular layer
50.0 m
11/39
GAT1-GFP expression in cerebellum:
basket cell terminals in molecular layer
50 m
12/39
A
B
C
The data suggest:
the intracellular GAT1-GFP
untreated boutons
translocation treatment
beads
D
Relative fluorescent intensity (% )
120
(31-33% of GAT1 in WT; 80%
in mGAT1-GFP)
is so close to the membrane
100
that it appears membrane80
bound in the confocal
60
microscope.
0.5 M
40
20
0
-1.2 -0.8 -0.4 0.0
0.4
0.8
1.2
1.6
Distance (  m )
13/39
Schematic drawing of a chandelier cell
based on immunostaining for parvalbumin
in human neocortex
Ch terminals
from Felipe et al, Brain (1999) 122, 1807
14/39
Calibrations:
Transparent 90-m dia beads with calibrated surface densities of EGFP
various
[His6GFP]
Incubate Ni-NTA beads
with measured
numbers of
His6 EGFP molecules
Include beads with EGFP-labeled cells
in the fluorescent microscope
15/39
Single-molecule fluorescence at increasing GFP densities
16/39
10
Intensity (counts/m *s)
40
2
35
Frequency
30
25
20
15
10
5
6
10
5
10
4
10
0
0
Measured/expected intensity
10
10
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
10
Macroscopic measurements
*
Slope = 1
*
7
3
2
10
50 100 150 200 250
Intensity (counts/GFP•s)
0
1
2
3
4
10
10
10
10 2 10
Expected density (GFP/m )
10
-11
10
-10
10
-9
10
-8
10
5
-7
[His6-GFP], M
Single-molecule and macroscopic
measurements of GFP density
10
1
10
2
10
3
10
2
Expected density (GFP/ m )
4
- absolute calibration
- accurate within 20%
- over 4 orders of magnitude
- useful on both wide-field and
confocal microscopes
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Molecular layer of cerebellum
Total GAT1/bouton: 9000
Volume density 5000 GAT1/m3,
Surface density 1340 GAT1/m2.
Axons: 640 GAT1/m
Cartridge in cortex
contains 365,000 GAT1
Pinceaux in cerebellum
7.8 million GAT1,
at a volume density of 7700
GFP/m3
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Nearly Complete Nicotinic Acetylcholine Receptor, a Well-Studied Cys-loop Receptor
~ 2200
amino acids
in 5 chains
(“subunits”),
Binding
region
MW
~ 2.5 x 106
Membrane
region
Colored by
secondary
structure
Colored by
subunit
(chain)
Cytosolic
region
(incomplete)
19/39
Upregulation as a thermodynamic consequence of nicotine-receptor Interactions
Nicotine accumulates in cells
1 M Nicotine+
(pKa = 7.9)
Binding eventually favors high-affinity states
+
unbound
0 mV
Free Energy
-70 mV
Bound
states
with
increasing
affinity
15 M Nicotine+
Highest affinity
bound state
C
AC
A2C
A2O
AD
Coordinate
Fluorescence assays for receptor changesReaction
in response
to chronic2 nicotine?
+
+
Free Energy
Free
subunits
Increasingly
stable
assembled
states
+ nicotine
RLS
Covalently
stabilized
RHS
Degradation AR*HS
?
Nicotine
Increased
High-Sensitivity
Receptors
Nicotine may stabilize subunit interfaces
Reaction Coordinate
0
20
40
60
hr
20/39
Candidate fluorescently tagged a4 and b2 subunit constructs
a4 nAChR
1 aa
629 aa
Ligand-binding domain
HA tag
1 aa
501 aa
M1 M2 M3 IC loop M4
c-myc tag CFP
YFP
a4-YFP-N1
b2 nAChR
b2-CFP-C
XmaI (34 aa)
HA tag YFP HA* tag
a4-YFP-N2
c-myc tag CFP
XmaI (34 aa)
b2-CFP-M
PPUMI (381 aa)
HA tag YFP
a4-YFP-M
BStEII (426 aa)
We avoided targeting and signaling motifs
a4 M3-M4 IC Loop
b2 M3-M4 IC Loop
VHHRSPRTH TMPAWVRRVF LDIVPRLLFM
360
VHHRSPTTHT MAPWVKVVFL EKLPTLLFLQ QPRHRCARQR
360
KRPSVVKDNC RRLIESMHKM ANAPRFWPEP ESEPGILGDI
400
400
CNQGLSPAPT FCNRMDTAVE TQPTCRSPSH KVPDLKTSEV
YFP
EKASPCPSPG SCHPPNSSGA PVLIKARSLS VQHVPSSQEA
440
LRLRRRQRER EGAGTLFFRK GPAADPCTCF VNPASMQGLA
CFP
GAFQAEPAAA GLGRSMGPCS CGLREAVDGV RFIADHMRSE
480
DDDQSVREDW KYVAMVIDR
459
AEGSIRCRSR SIQYCVSQDG AASLTESKPT GSPASLKTRP
520
SQLPVSDQTS PCKCTCKEPS PVSPITVLKA GGTKAPPQHL
560
PLSPALTRAV EGVQYIADHL KAEDTDFSVK EDWKYVAMVI
600
DR
602
440
PKA
PKC
Tyrosine kinase
Casein kinase
Calmodulin dependent kinase II
Cyclin dependent kinase 5
VHHRSPRTH TMPAWVRRVF LDIVPRLLFM
360
VHHRSPTTHT MAPWVKVVFL EKLPTLLFLQ QPRHRCARQR
360
KRPSVVKDNC RRLIESMHKM ANAPRFWPEP ESEPGILGDI
400
400
CNQGLSPAPT FCNRMDTAVE TQPTCRSPSH KVPDLKTSEV
YFP
EKASPCPSPG SCHPPNSSGA PVLIKARSLS VQHVPSSQEA
440
LRLRRRQRER EGAGTLFFRK GPAADPCTCF VNPASMQGLA
CFP
GAFQAEPAAA GLGRSMGPCS CGLREAVDGV RFIADHMRSE
480
DDDQSVREDW KYVAMVIDR
459
AEGSIRCRSR SIQYCVSQDG AASLTESKPT GSPASLKTRP
520
SQLPVSDQTS PCKCTCKEPS PVSPITVLKA GGTKAPPQHL
560
PLSPALTRAV EGVQYIADHL KAEDTDFSVK EDWKYVAMVI
600
DR
602
440
Ubiquitination motifs: K
Endocytosis motifs: YXXf, f = M, L, F; LL
ER retention motifs: R/K-X-R/K, R/K-R/K
Amphipathic helix contains ER export motifs:
Ds and Es
Raad Nashmi
21/39
A collection of fluorescent Cys-loop receptor subunits
α4
aa 1
M3 - M4 aa 629
loop
M3-M4
loop
Ligand binding M1 M2 M3
M4
β2
aa 1
HA tag XFP
aa 501
c-myc tag XFP
β2-XFP-M
α4-XFP-M
FRET pairs
XFP =
Cerulean
mCerulean
ECFP
mECFP
EGFP
mEGFP
λ
EYFP
mEYFP
Venus
mVenus
mCherry
m = monomeric
Analogous functional but less extensive fluorescent series
α6
β3
C. elegans GluClα, Gluclβ
(dopaminergic neurons)
(selective neuronal silencing
22/39
There are 3C2 = 6 interesting genotypes
of
fluorescent nAChR a4 mutants
All phenotypes are viable
&
neo-deleted
YFP,
Leu9-Ala-YFP,
CFP
Raad Nashmi
23/39
From Roger Tsien’s notes
II. How is FRET detected and measured?
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
24/39
Older microscopes could image at only a few wavelengths, using interference filters
Lakowicz 13-13
25/39
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)
Lakowicz 13-13
26/39
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
27/39
FRET measurements by acceptor photobleaching (donor dequenching)
Intercept = 0.24
FRET efficiency =
1- (1/(1 + Intercept))
= 19.4%
0
80
20
40
60
YFP intensity decrease (%)
100
28/39
Fluorescent subunits tell us about nicotinic receptor assembly
*
Neuro2a
Ryan Drenan
29/39
Theory of FRET in pentameric receptors with αnβ(5-n) subunits
50% α-CFP, 50% α-YFP
a
a
a
a
a
a
1/4
1/2
1/4
E
No FRET
b/a =1.62; 1.62-6 = 0.055
a
a
a
a
a
1/8
1/4
1/8
1/4
a
E1
a
a
a
E2
E3
a
a
a
a
a
1/8
a
a
a
1/8
a
E4
No FRET
FRET Efficiency
60
100% (a4)2(b2)3
40
20
20
FRET efficiency
100% α3β2
100%
α2β3
100% (a4)3(b2)2
80
y = 0.1685x + 2.11
16
12
8
4
y = -0.1685x + 18.96
0
0
0
20
40
60
80
100
Distance between adjacent subunits, A
0
20
40
60
80
percent of 2-subunit receptors
100
% receptors with α3
30/39
Data:
Changes in subunit stoichiometry caused by varying subunit expression levels
20
18
18
16
16
% FRET Efficiency
% FRET Efficiency
14
12
10
8
6
14
12
10
8
6
4
4
2
2
0
0
4:1
1:1
1:4
ratio, a4CFP + a4YFP) : b2
1:9
1:9
1:4
2:3
4:1
9:1
ratio, a4 b 2CFP + b 2XFP)
Cagdas Son 31/39
The key experiment:
changes in subunit stoichiometry caused by chronic nicotine!
16
% FRET Efficiency
14
12
10
8
6
4
2
0
control
+ Nicotine
control
+ Nicotine
a4CFP + a4YFP) : b2
a4  b2CFP + b2YFP)
1:1
1:1
Cagdas Son 32/39
Fluorescence Intensity Ratio Analysis of Subunit Stoichiometry
(Hummer, Delzeith, Gomez, Moreno, Mark, & Herlitze (2003), JBC 278, 49386
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: a4X1FP, a4 X2FP at equal densities; b2X1FP, b2X2FP.
33/39
Fluorescence Intensity Ratios Also Yield Overall Subunit Stoichiometry:
a4/b2 = √(k1/k2)
cDNA: 19 ab
a4CFP b2YFP
a4YFP b2CFP
70
[Nic]
M
total
b2/a4
%
(a4)2(b2)3
0
1.08
70
1
1.45
95
50
60
40
CFP intensity
50
40
30
30
20
20
k2 = 0.166
50
40
30
20
10
slope = 0.1955
0
0
50
100
150
200
250
10
10
k1 = 0.196
0
0
0
100
200
300
0
400
80
800
60
600
40
400
50
100
k2 = 0.204
200
0
0
100
200
300
200
250
k1 = 0.425
20
0
150
400
0
500
1000
1500
YFP intensity
Cagdas Son 34/39
The in vivo Nonsense Suppression Method for Unnatural Amino Acid Incorporation
1
Gene for the protein
with “stop” codon at
site of interest
Inject into
Xenopus oocyte
mRNA or cDNA
STOP (“nonsense”) codon
(UAG = “amber”)
2
mix
“amber suppressing”
tRNA based on
Tetrahymena thermophila
a. Unnatural amino
acid chemically
appended
b. Appropriate
anticodon
OR
Ribosome
incorporates
unnatural
amino acid;
protein
reaches
membrane
(12-48 h)
Im
Vm
Functional
measurements on
the protein
containing the
unnatural
residue
Im / Vm
Electroporate into
cultured
mammalian cells
35/39
Lys-BODIPYFL: a fluorescent unnatural amino acid
4-base codon method
36/39
Lys-BODIPYFL: a fluorescent unnatural amino acid
37/39
Counting photobleach steps to assess stoichiometry
38/39
End of lecture
39/39