Characterization of protein folding intermediates for delineation of folding pathways

A solution to the protein folding puzzle---how amino acid sequence determines protein structure---will have huge impact in a variety of research areas ranging from protein design and production to aggregation-related diseases and disorders. The characterization of protein folding intermediates likely holds a crucial key to understanding protein folding mechanism. In spite of the countless folding intermediates which have been successfully studied, two major issues remain to be addressed: (1) The need of a general method to find and trap folding intermediates. (2) Lack of knowledge about protein folding at the early stages of the folding reaction.;Using ubiquitin as model protein, I developed a novel "Protein Vivisection" strategy that involves replacing buried hydrophobic residues with charged ones to partially unfold a protein. Using this strategy, I identified two folding intermediates which had been unobservable using previously available methods. Characterization of these intermediates led to full delineation of ubiquitin post-transition state pathway.;I then moved on to investigate the pre-transition state events of the ubiquitin folding reaction. Peptide fragments of ubiquitin were used as mimics of early folding intermediates to demonstrate (1) Secondary structure elements formed at early stage are stabilized by native-like tertiary contacts. (2) Marginally stable early folding intermediate may be identified by native-state hydrogen exchange data. Together, these results are consistent with the view that at early stages of folding, formation of secondary structure and native tertiary contact is concerted and there is a preferred pathway for folding to proceed.