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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

2/39

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

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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

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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?

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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

24/39

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

35/39

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

37/39

Lys-BODIPYFL: a fluorescent unnatural amino acid

38/39

Counting photobleach steps to assess stoichiometry

39/39

End of lecture