Figure 3.

Experimental low-temperature (100 K) FTIR spectra reveal a single nitrile population for F62oCNF but two populations for F92oCNF. Protein samples were buffer exchanged into glass forming 1:1 mixtures of glycerol and aqueous buffer to enable low-temperature studies. (A) F62oCNF’s spectrum displays a single band centered at 2230.8 cm^–1. At room temperature in the buffer, a single band was also observed (Figure S11).²⁴ (B) F92oCNF’s spectrum displays two bands (2230.1 and 2246.9 cm^–1 for minor and major peaks, respectively; fits are shown as black dotted lines). In contrast, the room temperature spectrum contains a single band at 2241.3 cm^–1 (Figure S11).²⁴ The apparent disparity in the number of IR populations at low and room temperature can be explained by rapid chemical exchange between the species at room temperature, as predicted by simulations. IR spectra for F62oCNF and F92oCNF (in addition to F28oCNF and F96oCNF) at additional temperatures up to 323 K can be found in Section S5.