Understanding the folding mechanisms of small model proteins

Most proteins require appropriate folding in order to perform their respective functions, whereas misfolding can lead to pathological conditions. Thus, protein folding presents a complex problem which requires extensive study from both physical and biological perspectives. While significant progress has been made towards a solution to the protein folding problem, a quantitative and predictive understanding of how proteins fold is yet to be reached. This is partly due to the fact that protein molecules are complex, and that many (weak) interactions work together to define a protein\u27s native structure. To reduce the complexity of the problem, we have taken a bottom-up approach and focused on studying the folding dynamics and mechanism of small peptides that exhibit folding characteristics of large proteins. These studies not only yield insights into the early steps of protein folding, including the theoretical folding speed limit, but they also provide ideal models for computer simulations. The systems described in this thesis include simple protein secondary structural elements and miniproteins (i.e., trp-cage, GCN4-p1, and villin headpiece subdomain). Since these peptides fold on the sub-millisecond timescale, we have employed a laser-induced temperature jump infrared technique to measure their folding kinetics. Whenever necessary, we have also made use of sequence and structure perturbing methods to systematically assess the mechanistic role of various native-state properties, including protein length, hydrophobic and electrostatic interactions, and backbone-backbone hydrogen bonding, in the formation of the transition state ensemble. For example, our study of Trp-cage folding indicates that, contrary to molecular dynamics simulation, the formation of a solvent exposed salt-bridge is not required for achieving fast folding; instead, a well-placed aromatic interaction lowers the folding free energy barrier. Also, through investigation of the folding dynamics of a GCN4 coiled-coil variant, we show that the folding of such dihelical structural motifs is likely initiated by contacts throughout the sequence rather than those localized in a previously identified trigger sequence. Furthermore, using amide-to-ester backbone mutations, we are able to demonstrate that helix formation is not necessary for acquiring the transition state in the folding of the helical subdomain of villin headpiece, discrediting the backbone-centered view of protein folding