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Chemical Methods for
Imaging Proteins in a Cell
Kimberly Peterson
Gellman Group
October 12, 2006
Complexity of Proteins in a Cell
• Proteins have numerous roles in biomolecular processes
– Enzyme catalysis of metabolic reactions
– Decoding and transmitting information
from DNA
– Building, transporting, and transforming
cellular components
– Interacting with each other in signaling
pathways
β2AR Signaling
http://www.biocarta.com/pathfiles/m_cftrPathway.asp
Voet, D.; Voet, J.G.; Pratt, C.W. Fundamentals of Biochemistry; John Wiley & Sons, Inc. : New York, NY, 1999; p 162
2
Labeling a Purified Protein Out of a Cell
• Amine-reactive fluorophores (Lys, N-terminus)
– ex. Fluorescein isothiocyanate (FITC)
• Sulfhydryl-reactive fluorophores (Cys)
= protein
– ex. Tetramethylrhodamine-5-iodoacetamide
Harmanson, G.T. Bioconjugate Techniques; Academic Press : Rockford, IL, 1996; p. 298-326.
3
Labeling a Protein in a Cell
• Components of an effective labeling technique
–
–
–
–
–
Specific interaction
Fast association
Protein unperturbed (structure, function)
General for any system
Non-toxic
Miller, L.W.; Cornish, V.W. Curr. Opin. Chem. Biol. 2005, 9, 56.
4
Fluorescence Overview
nm
400
400
500
500
600
600
700
700
http://en.wikipedia.org/wiki/
Electromagnetic_spectrum
Periasamy, A. Methods in Cellular Imaging; Oxford University Press : New York, NY, 2001; p. 6.
5
Methods of Imaging a Protein in a Cell
• Most general: autofluorescent proteins
• Smallest label: unnatural amino acids
• Compromise between two extremes
– Development of new techniques
– Applications
6
Green Fluorescent Protein (GFP)
History
• Discovered by Shimomura in the
jellyfish Aequorea victoria
• Gene sequenced 1994
• Crystal structure solved 1996
http://science.nasa.gov/headlines/y2001/ast01jun_1.htm
Structure
• 11-stranded β-barrel structure
– chromophore in center
• Molecular weight (MW) ~ 25 kDa
• λex = 395, 470 nm, λem = 509 nm
Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509.
Chudakov, D.M.; Lukyanov, S.; Lukyanov, K.A. Trends Biotechnol. 2005, 23, 605.
Zhang, J.; Campbell, R.E.; Ting, A.Y.; Tsien, R.Y. Nat. Rev. Mol. Cell Biol. 2002, 3, 906.
7
GFP Chromophore Biosynthesis
Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509.
8
GFP Chromophore Environment
Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509.
9
CFP Chromophore Environment
Tyr66 (GFP)  Trp (CFP)
Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509.
10
More Autofluorescent Proteins (AFPs)
Tsien, R.Y. FEBS Lett. 2005, 579, 927.
11
Fluorescence Resonance Energy
Transfer (FRET)
Donor + Acceptor
Fluorescence
of FRET pair
Periasamy, A. Methods in Cellular Imaging; Oxford University Press : New York, NY, 2001; p. 258.
12
Using AFPs as a Methylation Sensor
476 nm
527 nm
FRET
433 nm
433 nm
Methyltransferase
+
NH3
Histone
Peptide
Demethylase
+
NMe3
Methyllysine
Binding Domain
• First reported sensor for methylation of the N-terminal tail
of a histone protein
• Tested for methylation of Lys9 from the histone protein H3
Lin, C.W.; Jao, C.Y.; Ting, A.Y. J. Am. Chem. Soc. 2004, 126, 5982.
13
Specificity of the Methylation Sensor
70
Methylation
Site
mutant
% Emission
Ratio Change
60
50
Wild-type MEF
Suv39h -/- MEF
2.15
40
30
Binding
domain
mutant
20
10
1.00
0
Original
W45A
K9L
• YFP/CFP emission ratio change is specific for the binding of
methyllysine to its binding domain
• Works in mouse embryonic fibroblasts (MEF)
– Lower FRET in cells lacking a K9 specific methyltransferase
Lin, C.W.; Jao, C.Y.; Ting, A.Y. J. Am. Chem. Soc. 2004, 126, 5982.
14
Analysis of Labeling Techniques
GFP
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
15
Chemical Analogue of GFP –
Unnatural Amino Acids
• Selectively incorporate unnatural amino acids in a protein
during expression in a cell
Wang, L.; Schultz, P.G. Angew. Chem. Int. Ed. 2005, 44, 34.
Beatty, K.E.; Xie, F.; Wang, Q.; Tirrell, D.A. J. Am. Chem. Soc. 2005, 127, 14150.
Wang, J.; Xie, J.; Schultz, P. J. Am. Chem. Soc. 2006, 128, 8738.
16
First Amino Acid Fluorophore
Coumarin
Incorporated into Protein
A B
A B
A. control
B. mutant
Myoglobin
Wang, J.; Xie, J.; Schultz, P. J. Am. Chem. Soc. 2006, 128, 8738.
17
Analysis of Labeling Techniques
GFP
Unnat.
aa
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
18
Chemical Methods of Protein Labeling
Large
Autofluorescent Proteins
Antibody – Hapten Complex
hAGT Fusion
Size of Label
DHFR Fusion
Protein Splicing
Biarsenical – Tetracysteine Complex
Unnatural Amino Acids
Small
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Interaction of Protein with a Fluorescent Ligand
Antibody (sFv)
Hapten (phOx)
Fluorescein
• One of the first examples of labeling a protein in a
cell with a ligand-receptor complex
– Antibody MW ~ 28 kDa
– Measured Golgi pH based on fluorescence (sensitive to
environment)
Farinas, J.; Verkman, A.S. J. Biol. Chem. 1999, 274, 7603.
20
Analysis of Labeling Techniques
GFP
Unnat.
aa
Antibody
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
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Covalent Labeling via Protein Fusion
Natural function:
Labeling the protein to be studied:
hAGT = O6-alkylguanine-DNA alkyltransferase (MW ~ 20 kDa)
Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86.
22
First Label Design –
Benzylguanine (BG) Derivatives
Other Labels
Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86.
A.; Kindermann, M.; Gendreizig, S.; Pick, H.; Vogel, H.; Johnsson, K. Methods. 2004. 32, 437.
Keppler,
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Multi-Color Analysis of Vesicular Stomatitis
Virus Glycoprotein (VSVG)
Experiment Setup
VSVG
1. Label existing VSVG-AGT with
fluorescein at 34˚C
2. Quench residual AGT with O6(4-bromothenyl)-guanine
3. Raise temperature to 40˚C
(misfold new VSVG)
BG-SF
10 µm
O6-(4-bromothenyl)guanine
4. Label misfolded VSVG-AGT
with SF
A. Transmission image +
Fluorescence images
B. Fluorescein at plasma
membrane
C. SF in internal membrane
structures
Keppler, A.; Pick, H.; Arrivoli, C.; Vogel, H.; Johnsson, K. Proc. Nat. Acad. Sci. 2004, 101, 9955.
24
Increasing Substrate Specificity
Arg 135
Ser145
Inhibitor
Met 134
Gly 132
Gly 131
O6-methylguanosine bound to hAGT
• Goal: label mutant hAGT selectively over wild type (wt) hAGT
– Initial studies used hAGT deficient cells
• Method: add inhibitor to block wt hAGT active site
Juillerat, A.; Heinis, C.; Sielaff, I.; Barnikow, J.; Jaccard, H.; Kunz, B.; Terskikh, A.; Johnsson, K. ChemBioChem.
2005, 6, 1263.
25
Testing Selectivity of CG Inhibition in Cells
no
inhibitor
BG
CG
General
substrate:
MAGT-β-Gal
WTAGT-NLS
3
MAGT-β-Gal
+
WTAGT-NLS
Inhibitor:
3
β-Gal (protein)  cytoplasm
NLS (peptide)  nucleus
• CG enables selective labeling of mutant AGT (MAGT) in
the presence of wild type AGT (WTAGT)
Juillerat, A.; Heinis, C.; Sielaff, I.; Barnikow, J.; Jaccard, H.; Kunz, B.; Terskikh, A.; Johnsson, K. ChemBioChem.
2005, 6, 1263.
26
Analysis of Labeling Techniques
GFP
Unnat.
aa
Antibody
hAGT
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
27
Noncovalent Labeling via Protein Fusion
• Dihydroxyfolate reductase (DHFR)-Methotrexate (Mtx)
– DHFR = 18 kDa
• Initially used fluorescently labeled Mtx to quantify
amount of DHFR in cells
label
Mtx
DHFR
Protein
(Kd ~ 1 nm)
Kaufman, R.J.; Bertino, J.R.; Schimke, R.T. J. Biol. Chem. 1978, 253, 5852.
Miller, L.W.; Sable, J.; Goelet, P.; Sheetz, M.P.; Cornish, V.W. Angew. Chem. Int. Ed. 2004, 43, 1672.
28
Initial Studies with DHFR Fusions
Target
Plasma
membrane
Nucleus
Protein
Expressed
Label
CFP
-
DHFR
Mtx-Texas
Red (TR)
DHFR
Mtx, then
Mtx-TR
DHFR
Mtx-TR
Fluorescence
Transmission
Image
• Requires DHFR-deficient cells for specificity
Miller, L.W.; Sable, J.; Goelet, P.; Sheetz, M.P.; Cornish, V.W. Angew. Chem. Int. Ed. 2004, 43, 1672.
29
Advances with DHFR Fusions
• Selectively label E. coli (e)DHFR-protein fusion with
trimethoprim (TMP)
– KD for eDHFR = 1 nM, for mammalian DHFR = 4 µM
Miller, L.W.; Cai, Y.; Sheetz, M.P.; Cornish, V.W. Nat. Methods. 2005, 2, 255.
30
Selectively Label eDHFR Fusion
Target
CFP &
eDHFR
Protein
Expressed
Label
Plasma
membrane
TMP-BTR
Nucleus
TMP-BTR
Nucleus
TMP, then
TMP-BTR
Excitation
Wavelength
458 nm
568 nm
Transmission
Image
Miller, L.W.; Cai, Y.; Sheetz, M.P.; Cornish, V.W. Nat. Methods. 2005, 2, 255.
31
Analysis of Labeling Techniques
GFP
Unnat.
aa
Antibody
hAGT
DHFR
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
32
Protein Splicing in Nature
MW ~ 16.5 kDa
NT = N-terminus
CT = C-terminus
Noren, C.J.; Want, J.; Perler, F.B. Angew. Chem. Int. Ed. 2000, 39, 450.
33
Protein Splicing in a Lab
Creating a Protein with an
N-terminal Cys
Native Chemical Ligation
(NCL)
Muir, T.W. Annu. Rev. Biochem. 2003, 72, 249.
34
Labeling via Native Chemical Ligation
= label
• Selectively label protein with N-terminal Cys
– Proteins with an N-terminal Cys are rare in nature
Yeo, D.S.Y.; Srinivasan, R.; Uttamchandani, M.; Chen, G.Y.J.; Zhu, Q.; Yao, S.Q. Chem. Commun. 2003, 2870.
35
Thioester Labels
Coumarin
Ac-fluorescein
Tetramethylrhodamine
Biotin
Ac-naphthofluorescein
Caged fluorescein
Yeo, D.S.Y.; Srinivasan, R.; Uttamchandani, M.; Chen, G.Y.J.; Zhu, Q.; Yao, S.Q. Chem. Commun. 2003, 2870.
36
Analysis of Native Chemical Ligation Method
= EGFP
% EGFP labeling
• Labeling of purified protein over 24 h
Ac-fluorescein
Tetramethylrhodamine (TMR)
Ac-naphthofluorescein
Biotin
Time (min)
• Selective labeling of N-terminal Cys on
glutathione S-transferase (GST) with TMR
= GST
5 µm
Yeo, D.S.Y.; Srinivasan, R.; Uttamchandani, M.; Chen, G.Y.J.; Zhu, Q.; Yao, S.Q. Chem. Commun. 2003, 2870.
37
Analysis of Labeling Techniques
GFP
Unnat.
aa
Antibody
hAGT
DHFR
Splicing
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
38
Interaction of Cys Tag with Biarsenical Label
FlAsH = Fluorescent
Arsenical Helix Binder
Ethane dithiol
(EDT)
FlAsH-EDT2
α-helical peptide
Cys-Cys-Xaa-Xaa-Cys-Cys
Fluorescent
complex
Griffin, B.A.; Adams, S.R.; Tsien, R.Y. Science. 1998, 281, 269.
39
Proof of Principle – Selective Labeling
• CFP-Cys peptide expressed in HeLa cells, then treated
with FlAsH
– FRET observed between CFP & FlAsH
before
FlAsH
after 1 µM
FlAsH-EDT2
with excess
(1 mM) EDT
Transmission
Image
CFP
emission
480 nm
FlAsH
emission tail
635 nm
Griffin, B.A.; Adams, S.R.; Tsien, R.Y. Science. 1998, 281, 269.
40
Variety of Biarsenical Compounds
λex
λem
(nm) (nm)
Unique Properties
FlAsH
508
528
-
ReAsH
593
608
-
BArNile
520
604
Sensitive to polarity of
environment
522
4x brighter than FlAsH,
lower pH sensitivity than
FlAsH
544
FRET pair with F2FlAsH,
lower pH sensitivity than
FlAsH
F2FlAsH
F4FlAsH
500
528
Griffin, B.A. et al. Science. 1998, 281, 269.
Adams, S.R. et al. J. Am. Chem. Soc. 2002, 124, 6063.
Nakanishi, J. et al. Anal. Chem. 2001, 73, 2920.
Spagnuolo, C.C. et al. J. Am. Chem. Soc. 2006, 128, 12040.
41
Representative Syntheses of Labels
Griffin, B.A. et al. Science. 1998, 281, 269.
Adams, S.R. et al. J. Am. Chem. Soc. 2002, 124, 6063.
42
Optimizing the Tetracysteine Peptide
kon
CCXCC
X=
(M-1s-1)
peptide sequence
(one letter codes)
kexch
-1
(s )
4.5 x 10-6
Ac-WEAAAREACCRECCARA-NH
Ac-WEAAAREACCRECCARA-NH22
RE
Ac-WDCCPCCK-NH 2
Ac-WDCCPCCK-NH
P
Ac-WDCCPGCCK-NH
Ac-WDCCPGCCK-NH22
PG
100000 1.5 x 10-6
310000 1.2 x 10-6
Ac-WDCCGPCCK-NH
Ac-WDCCGPCCK-NH22
GP
50000
65000
3.6 x 10-6
Kd
(pM)
70
150
4
72
=
• Hairpin-like conformation (Pro-Gly) favored for tetracysteine
peptide
Adams, S.R.; et al. J. Am. Chem. Soc. 2002, 124, 6063.
43
Imaging Connexin Trafficking
Extracellular Space
Cx43 Cx43
Membrane
Cytoplasm
Tetracysteine
peptide (TC) +
ReAsH
FlAsH
Anti-Cx43
Antibody
• Six connexin43 (Cx43)
proteins assemble to form a
channel
– connect two cells through these
channels
Propidium
Iodide
Staining
Propidium Iodide - used to
highlight cell bodies and nuclei
5 µm
Gaietta, G.; Deerinck, T.J.; Adams, S.R.; Bouwer, J.; Tour, O.; Laird, D.W.; Sosinsky, G.E.; Tsien, R.Y.; Ellisman, M.H.
Science. 2002, 296, 503.
44
Pulse-Chase Analysis of Connexin Trafficking
Experiment Setup
1.
2.
3.
Label existing Cx43-TC with
FlAsH (green)
Wash away unbound label and
incubate 4 h
Label new Cx43-TC with
ReAsH (red)
A = stereopair confocal image
B = different size channels
C = asymmetry of two channels
in close proximity
2 µm
Gaietta, G.; Deerinck, T.J.; Adams, S.R.; Bouwer, J.; Tour, O.; Laird, D.W.; Sosinsky, G.E.; Tsien, R.Y.; Ellisman, M.H.
Science. 2002, 296, 503.
45
Monitoring the Conformation of β2AR
• Study β2 adrenergic receptor (β2AR) conformation using FRET
– FlAsH label not perturb structure of loop
• Conformational change occurs more rapidly than previous
measurements using purified protein
– Can help to clarify structure-function relationship
Nakanishi, J.; Takarada, T.; Yunoki, S.; Kikuchi, Y.; Maeda, M. Biochem. Biophys. Res. Commun. 2006, 343, 1191.
46
Analysis of Labeling Techniques
GFP
Unnat.
aa
Antibody
hAGT
DHFR
Splicing
FlAsH
Specific
Interaction
Fast
Association
Protein
Unperturbed
Generally
Applicable
Non-toxic
47
Conclusion
• Many chemical methods to label proteins in a cell
have been developed
– Enables study of a protein within its cellular
environment
– Can derive new information not accessible with studies
that use purified proteins in isolation
• Techniques are chosen based on the protein to
be studied and the analysis desired
– Balance between specificity and size
48
Future Direction
• Utilize existing methods to further probe cellular
processes
– Expand from a proof of principle to a routinely applied
technique
• Image proteins at low expression levels
49
Acknowledgements
• Professor Sam Gellman
• Gellman Group
• Additional Practice Talk Attendees
– Joe Binder
– Amanda King
– Jenny O’Neill
– Katie Partridge
– Kristin Plessel
50