1. Ultrafast studies of resonance energy
transfer in myoglobin: A-helix and local
conformational fluctuations
J.A. Stevens, J.J. Link, Y.-T. Kao, 
C. Zang,  L. Guo, and D. Zhong
Department of Physics
The Ohio State University
Ultrafast studies of resonance energy transfer in myoglobin: A-helix and local conformational fluctuations
J.A. Stevens, J.J. Link, Y.-T. Kao, 
C. Zang,  L. Guo, and D. Zhong
Myoglobin (Mb), a heme containing protein, is involved in the storage and release of ligands. We report here our studies of resonance energy transfer in Mb using an intrinsic tryptophan (Trp) and the prosthetic heme as an energy transfer pair. With site-directed mutagenesis, we placed one-at-a-time a single Trp donor into four locations on the A-helix. Utilizing the femtosecond up-conversion method, we examined a series of energy transfer dynamics in Mb. A molecular dynamics (MD) simulation was also used to infer structure and dipole orientation fluctuations for specific Trp. Both methodologies were used to characterize the local dynamic nature of Mb in solution compared to the static crystal structure

2. Sperm Whale Myoglobin PDB:1JP6 Heme W7 W14 W15 W12
Heme containing protein involved in the storage and release of ligands (O2,CO)
Model system
hydrogen atom:physics::
myoglobin:biochemistry
Investigate structure in solution “real-time” with precise temporal & spatially resolution
ultrafast fluorescence upconversion
tryptophan(Trp)-Heme resonance energy transfer (RET)
site-directed mutagenesis
PDB:1JP6
Heme W7
mention: consists of 8 alpha helices and a prostheic heme group
W14
W15
W12

3. Femtosecond Upconversion setup
Zhong group
Can reconstruct time-resolved fluorescence at a single wavelength
Different crystal orientations for each wavelength

4. Resonance Energy Transfer (RET)
Excited State
hydration is λ dependant
Heme
hvpu
hvfl
khydration
+ kRET
kRET – Rate of energy transfer
kD – Rate of donor fluorescence w/o acceptor
fd – Quantum efficiency of donor probe
Jda – Integral overlap of donor emission
with absorption of acceptor
Structural insight from:
r – Distance from donor to acceptor
κ2 – Orientation between transition dipoles
of donor and acceptor W7 kD
Intermediate ki
Describe that we directly monitor the fluorescence decay dynamics, which can be utilized to determine the radiationless energy transfer. To measure the fluorescence decay on such an ultrafast time scale, we implement a nonlinear optical technique named femtosecond-resolved fluorescence up-conversion.
Protein
Ground State
Choose 330nm
Without Energy Acceptor: 2.8 ns
With Energy Acceptor: ps 4

5. RET for 4 mutants on the A-helix
Trp
τRET (ps)
W12
212
W15
158**
W14
111 W7 22
** average RET time, may suggest multiple conformations for W15

6. Temperature dependence of W7 & W14 RET
RET: rate increases as T increases
Near beginning of A-helix, region is likely more flexible
W14
RET: no change with T
Buried inside protein, more rigid
W7: What does it change to?

7. Orientation Factor (κ2)
38º
1La
Trp
Donor:
Depends on physical orientation of molecules
Can determine κ2 with atomic coordinates
Orientation of Heme & Trp transition dipoles known αD αA
αDA D T A
Acceptor:
50º
Heme θ
10º
Explain fish plot, Heme transition dipole, function of theta maybe make kappa squared bigger and theta, put a theta on the heme picture
Tryptophan:
Has 2 Transition Dipoles which are perpendicular, 1Lb and 1La, 1Lb immediately swithes to 1La
1La Transition Dipole is known to be at 38 degrees from the axis shown
Heme:
The transition Dipole is know to be measured found from the alpha-gamma-meso of the phorphyrin ring Literature has the axis from Degrees
From the xstal structure, we can determine the orientations of the transition dipoles with respect to each other. Using this information, kappa squared can be determined
Here are the plots of Kappa squared as a function of theta, where theta is the angle from the alpha-gamma meso axis on the heme
Normal and Disordered refers to the appropriate graph where Normal is shown. Most of the protein studied is in the Normal form, Dissordered can be formed with a chemical process but then switches back to Normal.
These are the values that we expect to get from the various methods on calculating kappa squared. To get the experimental value, we must know the precise rate of RET. In order to experimentally determine this rate, other rates must also be determined that are on the same order of magnitude. Only when these rates are determined can we find the precise RET rate. One such rate is solvation.
Gryczynski, Z.;  Lubkowski, J.;  Bucci, E. Methods in Enzymology 1997, 278, 
Andrews, D. L.;  Demidov, A. A.,  Eds.; Resonance Energy Transfer; John Wiley and Sons: Chichester, 1999.

8. κ2 : molecular dynamics, Xtal structure & experiment
Li, T.;  Hassanali, A. A.;  Kao, Y. T.;  Zhong, D.;  Singer, S. J. J. Am. Chem. Soc. 2007, 129, 

9. Conclusions κ2: consistent among Xtal, MD, & experiment
W14 is buried inside the protein & rigid
RET rate no correlation with temperature
κ2: consistent among Xtal, MD, & experiment
W7 is likely more flexible; near the beginning of structure
RET rate increases with temperature
κ2: consistent among Xtal and MD; strikingly different from experiment
Xtal structure may not give accurate representation of true flexibility of protein
W15
RET rate is explained w/stretch model, may suggest multiple conformations of Trp

10. Acknowledgments Prof. Dongping Zhong Lijuan Wang Prof. Justin Link
Ya-Ting Kao
Chen Zang
Dr. Lijun Guo
Zhong Group
Funding
National Science Foundation
Packard Foundation