Many biological systems require oligomerization of protein subunits to form an active enzyme. Why do protein subunits oligomerize to an active superstructure without the incidence of further aggregation to a protein precipitate? Further elucidation of the mechanisms of protein folding/oligomerization is required to understand the basic mechanisms of illnesses such as Alzheimer's Disease, Parkinson's Disease, and diabetes mellitus. Phycocyanin is an ideal protein system for studying oligomeric structure formation; it contains tetrapyrrole chromophores that can be used to report the quaternary structure of the protein. Specifically, we will use phycocyanin as a model system to understand the kinetics of quaternary structure formation in alpha-helical, water-soluble proteins. We will test a specific hypothesis found in the literature: is a 'structured' intermediate needed for the formation of oligomers? An intermediate with tertiary structure implies that there is a specific recognition site on the subunits; is it possible that the subunits associate earlier in the folding process using nonspecific hydrophobic interactions and then undergo further rearrangement to the active structure? We are also interested in determining the role of the covalently bound chromophore in the early part of the protein folding process; does the chromophore provide a nucleation site for protein folding? Or does the chromophore binding pocket form first? The following specific aims are proposed. First, the kinetics of the refolding reaction from urea denatured protein to active oligomer will be measuring using a stopped flow mixing chamber. The kinetic traces obtained will be fit using exponential functions to determine lifetimes for each rate determining step in the process. The protein concentration dependence of each step will be used to assign it to either a folding step (first order kinetics) or oligomerization (higher order kinetics). Second, the temperature dependence of the refolding kinetics will be fully investigated and the results used for transition state calculations. Finally, a detailed mechanism for formation of oligomeric structure in our model system will be developed.
| Thoren, Katie L; Connell, Katelyn B; Robinson, Taylor E et al. (2006) The free energy of dissociation of oligomeric structure in phycocyanin is not linear with denaturant. Biochemistry 45:12050-9 |
