Figure 6.

Experimental discrimination of structural models of the T4^∗ folding intermediate. (a) Plot of the transfer free-energy difference between the I and U state (ΔΔG_tr,I-U) for every possible contiguously folded intermediate of T4^∗ to 1 M sorbitol. The axes represent the portion of the N-terminal or C-terminal residues unfolded in the intermediate. The values are shaded according to value as denoted by the key. (b) Shown is the same plot as (a), except for 1 M TMAO. Of note is that the values have higher magnitude, as expected from transfer free-energy models. (c) Shown are the experimental constraints on potential intermediates that are considered. Shaded in orange and red are intermediates that are consistent with the population changes in going from buffer to 1 M TMAO and 1 M sorbitol, respectively. The yellow diagonal bar is the dimensional contour-length-based constraint from the BHMM. Shaded in cyan are the intermediates consistent with all three constraints. These are all intermediates where the N-terminal domain is mostly unfolded and the C-terminal domain is primarily folded. (d) Potential structural models of intermediates. Color-coding is identical to Fig. 1a. (I) Depicted is the N-terminal subdomain that is consistent with the population constraint of TMAO (folded amino acids 60–72). (II) Depicted is the N-terminal subdomain portion along with C-terminal subdomain portion folded to be consistent with distance restraint (folded amino acids 96–108). (III) Depicted is an intermediate that has 50 amino acids of the N- and C-terminus unfolded. This intermediate is consistent with both population restraints. (IV) Depicted is the C-terminal subdomain structural model that is consistent with both constraints (C-terminus). This is the only intermediate that is consistent with all three pieces of experimental data. To see this figure in color, go online.