Dr. Joseph Knab

The primary goal of this dissertation was to elucidate and quantify the physical basis of protein flexibility at terahertz (THz) frequencies in two distinct systems: lysozyme and Photoactive Yellow Protein (PYP). To this end, I have probed these systems using the technique of Terahertz Time-Domain Spectroscopy (THz-TDS) in order to determine how the flexibility changes as a function of various conditions, such as changes in hydration, temperature, conformation and ligand binding. Measurements on lysozyme films as a function of ligand binding suggest a decrease in flexibility with binding. A procedure for making uniform protein films was established. Hydration dependent measurements on lysozyme films reveal a transition in the flexibility at the point where the first hydration shell is filled. Analysis of the dielectric response showed that the response cannot be solely attributed to relaxational effects, but one must include at least one resonant contribution. Temperature dependent measurements on lysozyme solutions show that the so-called dynamical transition for proteins is present in the terahertz dielectric response. Arrhenius plots of the imaginary part of the permittivity yield the activation energy for the system and this is shown to be dependent on frequency. Non-linear Arrhenius plots are obtained in some cases, perhaps indicating a different origin for the class of motions responsible for the transition. The final part of this dissertation concerns measurements made on PYP. A hydration transition was observed in the THz dielectric response as a function of hydration in PYP films, similar to that observed in the lysozyme films. This transition coincides with the point where the conformational equilibrium of the protein itself changes, as verified via UV/VIS spectroscopic measurements. These data are discussed in terms of an activated process. The THz response was also studied as a function of conformational state occupation between two photointermediate states. Static measurements indicated loss of flexibility in the photo-excited state. This is consistent with loss of tertiary structure that is also associated with this particular photointermediate. Time-resolved measurements were also performed, though the results from these measurements did not indicate any such change. This apparent contradiction could not be resolved and can be attributed to sample degradation or heating effects.

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