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Time Resolved Femtosecond Optical Studies of Heme Proteins Myoglobin and Cytochrome c

Stevens, Jeffrey Alan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1299604642

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Physics.
Our goal was to develop methods to study protein dynamics on the picosecond to nanosecond time scale. We used wavelength and femtosecond resolved fluorescence and absorbance spectroscopy coupled with site-directed mutagenesis to study the heme proteins myoglobin and cytochrome c and determine protein dynamics occurring on the picosecond to nanosecond time scale. The intrinsic amino acid tryptophan was used as a local optical probe and placed one at a time with site-directed mutagenesis that allowed us to achieve a spatial resolution down to a single amino acid. We systematically characterized resonance energy transfer (RET) between a single tryptophan and the prosthetic heme group in myoglobin to develop a novel energy-transfer pair as a molecular ruler in heme proteins to study local conformation fluctuations. Using site-directed mutagenesis, we placed a single tryptophan throughout myoglobin. We observed picosecond RET in tens to hundreds of picoseconds. We also characterized steady state parameters associated with RET allowing us to determine the orientation factor between tryptophan and heme and thus examine changes at different mutation sites revealing relative local structure flexibility and conformation fluctuations. Additionally, local environment relaxation occurs on similar time scales as the RET dynamics, resulting in a nonequilibrium dynamic process. We were able to determine the time scales of such nonequilibrium energy-transfer dynamics and elucidate the mechanism of the nonexponential RET dynamics caused by local dynamic heterogeneity and/or local environment relaxation. In cytochrome c, RET between the lone intrinsic tryptophan and heme was used to study protein folding/unfolding for various states (ferric, ferrous and CO-bound ferrous) of cytochrome c. We systematic studied heme dynamics and induced protein conformational relaxations in cytochrome c and myoglobin with femtosecond transient absorption (TA). A wide range of probing wavelengths from the visible to the UV was probed to unambiguously determine the protein dynamics of the ferric and ferrous state of cytochrome c. For cytochrome c in the ferrous state, the heme undergoes ligand bond breaking and rebinding process generating proteinquakes that perturb the local heme site and shake global protein conformation which was detected by the nearby lone intrinsic tryptophan. Cytochrome c in the ferric state has dynamics that mainly occur at the local site, including ultrafast internal conversion, vibrational cooling, and complete ground-state recovery with no global conformation relaxation observed. We expanded our studies by coupling site-directed mutagenesis of tryptophan at various locations throughout cytochrome c utilizing TA studies. Ligand photolysis from heme was used to generate proteinquakes perturbing the local heme site and shakes global protein conformation and was observed by several of the tryptophan mutations. Additionally, we observed the protein dynamics as a function of denaturing agent. The region that we studied was the pre-unfolding transition region; hence the protein was “softened.” In myoglobin, we similarly characterized the heme dynamics of the ferric, ferrous and CO-bound ferrous states. We attempted to use tryptophan as a local probe of protein dynamics for CO-bound myoglobin, but concluded that the perturbation created by CO photolysis was much smaller compared to the proteinquake generated in cytochrome c.
Dongping Zhong, PhD (Advisor)
Ralf Bundschuh, PhD (Committee Member)
Jay Gupta, PhD (Committee Member)
Justin Wu, PhD (Committee Member)
Physics
myoglobin; cytochrome c; time-resolved spectroscopy; protein dynamics

Recommended Citations

Citations

  • Stevens, J. A. (2011). Time Resolved Femtosecond Optical Studies of Heme Proteins Myoglobin and Cytochrome c [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299604642

    APA Style (7th edition)

  • Stevens, Jeffrey. Time Resolved Femtosecond Optical Studies of Heme Proteins Myoglobin and Cytochrome c. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1299604642.

    MLA Style (8th edition)

  • Stevens, Jeffrey. "Time Resolved Femtosecond Optical Studies of Heme Proteins Myoglobin and Cytochrome c." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1299604642

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.