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

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

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

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: 111 ps 4

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

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?

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 50-60 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, 538-569. Andrews, D. L.;  Demidov, A. A.,  Eds.; Resonance Energy Transfer; John Wiley and Sons: Chichester, 1999.

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, 3376-3382.

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

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