Time-resolved optical methods for the study of protein folding and conformation.

Studies of protein folding are necessary in order to understand how a one dimensional chain of amino acids becomes a functional three-dimensional protein. An understanding of the physical basis of this process can lead to the ability to predict protein structures from amino acid sequences and to predict how the environment affects protein folding. Laser pump-probe spectroscopy can monitor protein folding in real time on time scales from nanoseconds to seconds. This spectroscopy may be used to excite molecules and probe their conformationally sensitive excited states, transient absorption for example, or to change solution characteristics such as pH and temperature in order to trigger protein conformational changes. Comparisons were made between the tryptophan triplet state lifetimes determined by phosphorescence and by transient absorption for alkaline phosphatase and phosphoglycerate kinase. Differences found in lifetimes reported by the two techniques were due to the transient absorption of non-luminescent species. Thus, transient absorption may be used to study protein conformation and folding in cases where the phosphorescence signal is unmeasurable; additionally it provides a window unto tryptophan photochemistry. Experiments on early events in protein folding or unfolding allow direct measurements of the time scales and mechanisms of collapse from the random coil state or of the breakdown of the native structure. In these experiments, a laser pulse was used to abruptly change the solution pH or temperature in order to initiate protein folding or unfolding. pH jumps of 2 pH units were achieved. However, the ultra-violet absorption of the pH jumping dyes obscures intrinsic protein fluorescence and absorption; therefore, peptides or proteins used for such experiments must be labeled with extrinsic probes. Such probes must be able to reveal protein conformational changes without interfering in the protein folding process itself. Temperature jump experiments allow studies of protein unfolding due to thermal denaturation as well as protein folding from cold denatured states. Steady state measurements of the cold denaturation of apomyoglobin at various pHs and heat denaturation of ribonuclease T1 were conducted, a temperature jump apparatus was constructed and temperature jumps of 15-20$\sp\circ$C were demonstrated.PhDBiological SciencesBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/129992/2/9711968.pd