Ultrafast Dynamics of
Resonance Energy Transfer
in Myoglobin
Justin J. Link
Jeffrey A. Stevens
Ya-Ting Kao
Dongping Zhong
The Ohio State University
Department of Physics
General Prospective of RET to Study
Protein Dynamics
1948 ~ Theodor Förster’s paper puts RET on the map
Zwischenmolekulare Energiewanderung und Fluoreszenz
“Intermolecular Energy Migration and Fluorescence”
Annalen der Physik (2, 55-75)
1958 ~ Sperm Whale Myoglobin crystal structure solved by
Kendrew et al., Nature (Mar 8) 181 (4610):662-6
1971 ~ Protein Data Bank established at Brookhaven National Laboratory
Today ~ RET has proven to be a powerful tool used to study protein-protein
interactions, protein-DNA interactions, protein conformational changes,
reaction kinetics, and molecular motors all on diverse time scales
Justin J. Link
The Ohio State University
Department of Physics
Femtosecond Up-Conversion
Pump
PM
Probe
Sample
Time Delay
Non-Linear Crystal
Detector
Oseoghaghare Okobiah
Justin J. Link
The Ohio State University
Department of Physics
Resonance Energy Transfer
From Literature:
n – Index of refraction
τD – Donor lifetime
Can Accurately Calculate:
fd – Quantum efficiency of donor probe
Jda – Integral overlap of donor emission spectrum
with absorption spectrum of acceptor
kRET – From experimental fitting
Unknowns:
D
D
αD
αDA
αA
T
A
r - Distance from donor and acceptor
κ2 - Transition dipole orientation factor
Zhong, PNAS, January 8, 2002, 99, 13-18
Andrews, DL et al, Resonance Energy Transfer, John Wiley and Sons Ltd, Chichester, (1999).
Justin J. Link
The Ohio State University
Department of Physics
Sperm Whale Myoglobin (Mb)
Heme
W7
W14
1JP6
Justin J. Link
The Ohio State University
Department of Physics
κ
2
Theory
W14
38º
1JP6
1L
a
Trp:
N
N
D
1JP6
W7
50º
θ
10º
Heme:
D
NN
Z. Grycszynski et. al., Meth. Enzyme 278 (1997) 538-569
Justin J. Link
The Ohio State University
Department of Physics
Solvation Dynamics
Apo Mb
Normalized Fluorescence Intensity (a.u.)
W7 Native
360 nm
W7 Native
360 nm
340 nm
340 nm
330 nm
330 nm
320 nm
320 nm
315 nm
315 nm
310 nm
310 nm
305 nm
305 nm
S. K. Pal et al., J. Phys. Chem. B 106, 12376-12395 (2002)
0
Justin J. Link
10 20 30 40
Delay Time (ps)
The Ohio State University
50
0
50 100 150 200 250 300
Delay Time (ps)
Department of Physics
Apo vs. Holo
Heme
Normalized Fluorescence Intensity (a.u.)
Normalized Fluorescence Intensity (a.u.)
No Heme
Apo-W14
310 nm
330 nm
340 nm
0
20
40
60
80
Holo-W14
310 nm
330 nm
340 nm
0
Delay Time (ps)
Justin J. Link
20
60
40
80
Delay Time (ps)
The Ohio State University
Department of Physics
Normalized Fluorescence Intensity (a.u.)
Normalized Fluorescence Intensity (a.u.)
W14 vs. W7
Holo-W14
310 nm
330 nm
340 nm
τRET ~ 20 ps
0
20
40
60
80
Holo-W7
310 nm
330 nm
340 nm
τRET ~ 111 ps
0
80
Delay Time (ps)
160
240
320
Delay Time (ps)
NOTE: Different Time Scales
Justin J. Link
The Ohio State University
Department of Physics
Complete Results
W14
1JP6
1JP6
W7
D
0.76
N
N
NN
0.65 D
Trp
fd
J (cm3/M)
r (Å)
1JP6
D (ns) RET (ps)
n
R0 (Å)
κ2
κ2
Exp.
1JP6
W14 0.17 5.04x10-14 15.14
2.6
20
1.33
34.08
0.65 0.75
0.20 5.69x10-14 21.40
2.8
111
1.33
36.65
0.76 0.20
W7
Z. Grycszynski et. al., Meth. Enzyme 278 (1997) 538-569
Justin J. Link
The Ohio State University
Department of Physics
Future Work
T95
Further mutations will be
studied to further probe various
regions of Mb
Conformation dynamics will be
studied via photolysis of
ligands (CO, NO, O2)
Use RET as a tool to study
protein dynamics in other
heme proteins
A84
A74
A71
W14
1BZR
S. Kachalova et al., Science 284, 473-476 (1999)
Justin J. Link
The Ohio State University
Department of Physics