Figure 1. Defining the conformational landscape of p27-D2.

(A) Large effective refocusing field strength (ω_eff) and low temperature ¹H^N relaxation dispersion (RD) experiments reveal multi-state conformational exchange for the intrinsically disordered protein, p27-D2. Conformational exchange is sensed by many residues within p27-D2, which exhibit quantifiable exchange contributions ( $R_{\mathit{\text{ex}}}=R_{2,\mathit{\text{eff}}}^{\mathit{\text{low}}-{\omega }_{\mathit{\text{eff}}}}-R_{2,\mathit{\text{eff}}}^{\mathit{\text{high}}-{\omega }_{\mathit{\text{eff}}}}$; the dashed line indicates a R_ex value of zero). The greatest observed conformational exchange is observed for residues in p27-D2 within three aromatic clusters, including W60 (yellow), W76 (blue), and Y88 (red); the ¹H^N displaying the largest amplitude of motion is Q77 (green). (B) The observed RD monitors the change in the effective transverse relaxation rate (R_2,eff) as a function of ω_eff and reports that some residues undergo conformational exchange that has two kinetic phases. Solid lines correspond to global fits of all RD data to a thermodynamic model that describes nuclei which sense two separate kinetic phases. Data presented in A & B were collected at 274 K. (C) Plot of R_2,eff measured at low ω_eff (grey points) where a maximal contribution of microsecond exchange is detected as compared to the transverse relaxation rate due to the apparent intrinsic fast nanosecond exchange ( $R_{2,0}^{\mathit{\text{app}}}$; black points) across all residues. $R_{2,0}^{\mathit{\text{app}}}$ was determined from the fitted RD data and shows that large amplitude ¹H^N RD permits extensive sampling of R_ex by using high ω_eff which substantially quenches RD. The fast microsecond exchange detected in p27-D2 accounts for the elevated R_2,eff values and once these are taken into account, a flat, featureless $R_{2,0}^{\mathit{\text{app}}}$ profile is observed as expected for a prototypical disordered protein.