Proteins play vital roles in the proper functioning of all biological systems. It has become a consensus in the field of biochemistry that protein function, structure and dynamics are closely related, among which the dynamics holds the key to understand how protein structure ultimately determines its biological functions. This dissertation tries to thoroughly investigate protein conformation dynamics and local hydration dynamics at the most fundamental level with state-of-the-art femtosecond lasers and molecular biology techniques.
Conformational fluctuations occur on many timescales, yet it is the ultrafast dynamics that are still not well understood. 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. We choose horse heart cytochrome c (Cyt c) as a model system to investigate the local environmental conformational changes in the vicinity of both the tryptophan and heme. By utilizing 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, we observed, upon photolysis of the methionine-Fe bond, a proteinquake on the ultrafast regime.
We also studied protein hydration dynamics, whose understanding has far-reaching implications relating to protein plasticity and recognition, protein folding and aggregation, and enzyme catalysis. With site-directed mutagenesis, tryptophan as a local optical probe was placed at desired positions on peptide or protein surfaces to detect environment response using the femtosecond-resolved fluorescence up-conversion technique and reveal hydration dynamics with single-residue resolution. Systematic studies are first performed in small model systems, such as α-helix, β-hairpin, and a 20-residue globular protein of ptryptophan-cage. They clearly show that a certain ordered, rigid water network has been formed even for simple secondary structures. And finally, as a large and flexible globular protein, calmodulin was chosen to map the global surface hydration dynamics, in its calcium free, calcium binding and in complexation with a target protein states. A series of water-network relaxations and coupled protein structural fluctuations has been observed. It is also elucidated that the protein solvation dynamics is highly correlated with its own conformational dynamics.