Fig. 3.

NOESY crosspeaks between the active site Trp 59 H^Nε1 and nearby methyl groups in the V101I variant (A) and wild type FKBP12 (B) for the F2-F3 plane at the indole ¹H^Nε1 frequency from a 3D F1-filtered ¹H-¹³C-¹H NOESY spectrum of collected using a 200 ms mix time (black) is superimposed upon the reference 2D ¹H,¹³C HSQC spectrum containing all selectively enriched valine C^γ, leucine C^δ and isoleucine C^δ positions (green). The observable crosspeaks from the 3D NOESY spectrum of the V101I variant predominantly reflect intensities that are consistent with the canonical orientation of the indole ring. The crosspeak for Val 24 C^γ2 is 10-fold weaker in the V101I spectrum. This crosspeak would be unobservable for the canonical orientation of the indole ring and is consistent with a corresponding 10-fold decrease in the population of the perpendicular reoriented indole ring, relative to the wild type protein.