An isolated helix persists in a sparsely populated form of KIX under native conditions

NMR relaxation dispersion techniques were used to investigate conformational exchange of the three-helix bundle protein KIX under native conditions. These experiments provide site-resolved kinetic information about microsecond-to-millisecond time scale motions along with structural (chemical shift) information without requiring a perturbation of the equilibrium. All kinetic data are consistent with an apparent two-state transition between natively folded KIX and a partially unfolded high-energy state that is populated to 3.0 +/- 0.2% at 27 degrees C. By combining C-13- and N-15-based experiments that probe specific structural aspects, we show that the sparsely populated high-energy state displays a strong conformational preference. An isolated secondary structural element, C-terminal helix alpha 3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha 1 and alpha 2, are disordered and devoid of specific interactions. This high-energy state presumably represents the equilibrium analogue of a folding intermediate that is transiently populated in stopped-flow kinetic experiments.