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 (13)C- and (15)N-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 alpha3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha1 and alpha2, 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 [Horng, J. C., Tracz, S. M., Lumb, K. J., and Raleigh, D. P. (2002) Biochemistry 44, 627-634].
Tollinger
Martin
M
Department of Biomolecular Structural Chemistry, University of Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria. martin.tollinger@univie.ac.at
Kloiber
Karin
K
Agoston
Bianka
B
Dorigoni
Cornelia
C
Lichtenecker
Roman
R
Schmid
Walther
W
Konrat
Robert
R
D050882:CREB-Binding Protein / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding
D050882:CREB-Binding Protein