Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology.

The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed.

These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.