An Isolated Helix Persists in a Sparsely Populated Form of KIX under Native Conditions

Abstract

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].

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

“…In general populations as low as 0.5 % can be assessed and studied by means of CPMG RD (Korzhnev and Kay 2008;Mittag and Forman-Kay 2007;Sugase et al 2007). However, it should be noted that up to today conformational transitions of IDPs, i.e., conformational sampling, taking place on the detectable time regime by CPMG at ambient temperatures were only rarely reported (Neudecker et al 2006;Tollinger et al 2006). Although CPMG is a promising alternative for the characterization of transient conformational sub-states, IDPs frequently sample their accessible conformational spaces too fast in order to be detected by means of RD.…”

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

“…The average value of the half-life is 11 h. The intermediate was found to be unexpectedly stable when compared with typical folding intermediates with much shorter half-lives on the microsecond-to-millisecond timescale. 3,[5][6][7][8][9][10][11][12][13] These results indicate that the transition from the intermediate to the native state is highly cooperative and requires gross structural rearrangement. (Table 1), compared to typical folding intermediates.…”

“…(Table 1), compared to typical folding intermediates. 3,[5][6][7][8][9][10][11][12][13] In many cases, slow folding reactions are caused by cis/ trans isomerisation of proline residues; that is, for proteins containing cis-proline in its native state, the trans → cis isomerisation slows the folding down because the trans isomer is more favoured in the unfolded state. [29][30][31] However, in the case of SBD, all five proline residues adopt the trans conformation in the native state.…”

“…3,[5][6][7][8][9][10][11][12][13] In some cases, it has been indicated that appropriate packing in the hydrophobic core of proteins is critical for efficient folding; insufficient burial and packing of core residues lead to the accumulation of folding intermediates. [10][11][12][13][14][15][16][17] In other cases, it has been shown that interactions formed on protein surfaces are important for efficient folding. 18,19 The polypeptide fragment containing the starchbinding domain (SBD) of Aspergillus niger glucoamylase is a small globular protein consisting of 110 amino acid residues.…”

Reassessing the folding of the KIX domain: Evidence for a two‐state mechanism

“…[14][15][16][17] The folding and unfolding reactions of KIX have been studied both by equilibrium and by kinetic experiments. 18,19 The analysis of the amplitude and rate constants obtained by refolding experiments suggested the KIX domain to fold via a low-energy intermediate, which accumulates in the dead time of the stopped flow. Because of the low stability of such an intermediate, it is unclear whether this species is the same as that detectable by equilibrium experiments.…”