Proteins are dynamical in nature. Their ability to function relies on their overall flexibility. Conformational fluctuations occur on many timescales yet, it is the ultrafast dynamics that are still not well understood. This dissertation is a systematic investigation of ultrafast protein conformational dynamics studied with ultrafast laser spectroscopy coupled with site-directed mutagenesis. Using a single intrinsic tryptophan as the local optical probe, we strived to develop optical techniques that would allow one to see in real time ultrafast protein conformational dynamics. Two methods were explored, each viewing the conformational dynamics in a different light. The first method involved resonance energy transfer (RET) between the known energy transfer pair of tryptophan and heme in proteins. RET is highly dependent upon the orientation and distance between the tryptophan and heme, thus, obtaining information of the RET characteristics will allow us to view relative distance changes between different conformations of proteins. The other method explored investigates the local environmental conformational changes in the vicinity of both the tryptophan and heme. This method utilizes ultrafast transient absorption to observe the absorption change of the ground-state of heme or tryptophan due to environmental response of the protein after perturbation, also known as the Stark effect.
Sperm whale myoglobin was chosen as the first model system to study due to the wealth of knowledge available. It also only contains two intrinsic tryptophans and one prosthetic heme, both of which are chromophores. Site-directed mutagenesis was used to create over 20 single tryptophan mutations in an effort to globularly map out the ultrafast conformational dynamics.
Characterization of the RET in mutants of different oxidation states was first completed using fluorescence up-conversion. Conformational dynamics were induced via photodissociation of CO from myoglobin and studied via both fluorescence up-conversion and ultrafast transient absorption. The former involved the implementation of an innovative 3-beam pump probe experiment where the latter utilized visible pump with UV-visible probing. The ultrafast transient-absorption was used in an effort to observe the ground-state tryptophan response and local heme environmental response upon photolysis of carboxy-myoglobin. The ultrafast conformational fluctuations upon ligand dissociation were determined to be extremely small and the mutation points of tryptophan were not sensitive to the fluctuations in myoglobin.
The second model system studied for the ultrafast conformational dynamics of proteins was the well studied horse heart cytochrome c (Cyt c). Similar studies were performed and we observed, upon photolysis of the methionine-Fe bond, a proteinquake on the ultrafast regime. The ground-state absorption change of tryptophan was observed using ultrafast transient-absorption. Upon the success of the experiment, 11 single tryptophan mutations were created in the hope to globularly map out the ultrafast conformational dynamics. Unfolding experiments on Cyt c were also studied by fluorescence up-conversion in efforts to elucidate the initial steps from unfolded to folded structure. Characterization of the unfolding curve was complete for several redox states of Cyt c using resonance energy transfer.