| Protein folding is the final step in the Central Dogma of biology whereby newly translated proteins achieve their unique three-dimensional structures. With the goal of determining the general biophysical laws that govern the process of protein folding, I rely on traditional methods while developing several novel experimental techniques. First, the rule of two-state folding for small globular protein is investigated by testing known exceptions to this rule. The exceptions to the phenomenon are often mistakenly due to protein aggregation, misfolding, solvent dependent relaxation, or multiple mismatched data sets. Therefore, the Initial Barrier Hypothesis is maintained for nearly all small globular proteins, which asserts that the major barrier to protein folding is limited by a search process and intermediates more stable than the unfolded state do not appear prior to this barrier. Second, a novel isotope effect measurement of backbone amide hydrogen bonds is developed. An average hydrogen/deuterium equilibrium isotope effect of 8.6 cal/mol/bond is assessed only for bonds in helical geometries. The number of helical hydrogen bonds that form in the transition state ensemble is determined using the kinetic partitioning of the isotope effect. Hydrogen bonds and solvent accessible surface area are buried concomitantly. It is hypothesized that the condensation of hydrophobic side chains excludes water from hydrogen bond partners, subsequently driving their formation. Third, a novel method, whereby bi-histidine metal ion binding sites are engineered into proteins, assesses whether multiple folding pathways exist by implementing psi-analysis. Psi-analysis resolves the limitations of traditional mutational phi-analysis in that both the proportion of molecules traversing a given pathway and fractional formation of a given site are assessed. Fold topology is found to play a crucial role in governing the degree of pathway heterogeneity for a protein. |
