Understanding protein folding is crucial in both the prediction of protein structure from sequence and the design of novel functions. With the recent completion of multiple genome projects, much effort is now focused on the determination of protein structure and function. The long-term goal of this proposal is to understand how proteins fold. The last granting period witnessed the development of several new methods to investigate protein folding. These advances place us in a unique position to address a series of fundamental questions in the kinetics of protein folding. Our first aim, utilizing kinetic isotope effects and specifically designed alpha-helical coiled coil variants, is to determine when hydrogen bonds from in the folding pathway and to test our hypothesis concerning the correlation between H-bond formation and surface burial for different protein types. Our second aim, utilizing designed metal binding sites to stabilize particular regions of a protein, is to investigate whether there are single or multiple pathways (e.g. funnels) as well as the relative importance of short and long-range contacts on folding pathways. Our third aim, utilizing hydrogen exchange/NMR techniques for the measurement of microsecond folding rates under native conditions, is to investigate how fast a protein can fold and to identify the ultimate limiting processes. Our multi-pronged approach applied to different structural classes will provide a comprehensive picture of how proteins adopt their biologically active structures. Our fourth aim expands our focus to the kinetic determinants of protein folding when it is coupled to DNA binding. The goal is to learn how proteins expeditiously find a particular nucleotide sequence in a cellular context. Folding experiments using methodologies outlined above with the DNA binding domain of transcriptional activator GCN4 will extend our understanding of protein/DNA recognition beyond the static and into the kinetic realm. These results will have general implications for many dynamic cellular processes.