The overall goal of this proposal is to elucidate the folding mechanisms of a family of (beta/alpha)8 or TIM barrel proteins, one of the most common structural motifs in biology. Our focus on this family of proteins stems from our interests in the relative roles of sequence and motif in defining the energy surface over which the folding reaction flows. A variety of biophysical techniques will be employed to understand how the folding landscape is influenced by non-random structures in the denatured state, by micro- to millisecond folding events, and by subsequent rate-limiting reactions. The hypothesis, that strings of non-polar amino acids often found in beta strands preferentially associate in highly denaturing solutions, will be tested by combining mutational analysis with small angle x-ray scattering (SAXS), Forster resonance energy transfer (FRET) and NMR/spin label measurements. The development of specific secondary and tertiary structure in the very early folding events will be examined with a novel continuous-flow (microseonds) and conventional stopped flow (milliseconds) mixing systems interfaced to FRET and far-UV CD detection systems and with a conventional quenched-flow hydrogen exchange (HX) system interfaced to mass spectroscopy (MS) detection methods. Mutational analysis of the rate-limiting folding reactions will yield structural insights into the process by which the rapid pre-organization of beta alpha modules is linked to the final conversion of the equilibrium intermediate to the native conformation. The folding mechanisms of several other non-classical (beta alpha)n barrel motif proteins will also be examined in an effort to define the generality of the folding mechanisms for proteins containing repeating beta alpha modules. Collaborative efforts will enable us to compare our results on partially-folded forms and the barriers that define them with the predictions from simulations based upon Go-like potentials. The interplay between experiment and computation will serve to validate this simplified computational approach and, possibly, allow us to obtain detailed structural information about key folding intermediates. The insights obtained from all of these studies are expected to have a significant impact on biochemistry, medicine and biotechnology.