Table 1.

Methods for characterizing the folding TS and levels of 2°-structure formation

Method	Observation
Guinn et al. analysis (2): Amide and hydrophobic surface burial	On average, 77% and 60% of the total amide and hydrophobic surface are buried in the TS, respectively; the level of amide burial is consistent with native 2°-structure formation
ϕ analysis: Energetic effects of altering side chains	TSs generally lack some 2° structures, particularly in polarized TSs
Ψ analysis: Side chain-side chain contacts	Large with extensive amounts of 2°-structure and native-like topology, but with some unstructured regions
H/D amide isotope effects: backbone hydrogen bond formation	Fraction of (helical) hydrogen bonds correlates to surface burial in the TS, typically with 50–90% of the hydrogen bonds formed
HX, RD: structure of post-TS hidden intermediates	Hidden intermediates often lack some 2° structures and place an upper bound on structure in the TS