Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding

Average area buried upon folding & helix propensities.

<p>Plot of the average area buried upon folding (AABUF; upper part) and the helix propensities (lower part) for TC5b (black) and its P12W (red) point mutant.</p

NMR-derived native state structure of TC5b (PDB entry: 1L2Y).

<p>Highlighted is the Trp 6 residue (blue) (see text) together with the cage-forming side chains of Tyr 3 (dark blue), Ile 4 (yellow), Leu 7 (light blue), Pro 12 (red), Arg 16 (green), Pro 18 (brown), and Pro 19 (pink).</p

Cluster analysis of denatured TC5b.

<p>Plot of the experimental ¹⁵N <i>R</i>₂ values (black line) and the results of the cluster analysis as described in the text (red line) against the residue number for 6 M urea-unfolded (<b>A</b>) TC5b and (<b>B</b>) its P12W mutant. Moreover, the dashed line (blue) represents the intrinsic ¹⁵N <i>R</i>₂ relaxation rates of the polypeptide as described by the first term of Eqn. 1.</p

Experimentally verified inter-residue contacts between Trp 6 and aliphatic side chains.

1<p>H-¹H NOE cross-peaks of the TC5b all-<i>trans</i> Pro isomer denatured in 6 M urea. Shown are all detected and assigned sequential and non-sequential NOE contacts between side chain ¹H nuclei of Trp 6 (bold) and protons of those side chains found to be associated with the tryptophan residue in the chemically denatured state; contacts involving H^N, H_α, and/or Trp H_β protons cannot be seen in this region of the spectrum. The numbers shown in brackets correspond to the assignment scheme of the respective NOE cross-peak as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041301#pone-0041301-g004" target="_blank">Figure 4</a>.</p

Reduced spectral density functions.

<p>(<b>A</b>) , (<b>B</b>) and (<b>C</b>) for 6 M urea-denatured TC5b derived from the 60.25 MHz <i>R</i>₁, <i>R</i>₂, and heteronuclear NOE relaxation data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041301#pone-0041301-g003" target="_blank">Figure 3</a>. The average errors are 2.5%, 1.2% and 2.7%, for , and , respectively.</p

Assigned main chain contacts between H^N and/or H_α nuclei.

<p>Assigned main chain NOE cross-peaks involving either H^N and/or H_α nuclei for 6 M urea-denatured TC5b. Residues found in the helical regions of the native mini-protein are shown in bold. An NOE between the H^N proton of residue and the H^N proton of residue is marked as a black square, NOEs between the H_α of residue and the H^N of as a filled triangle, and an NOE between any atom of the side chain of residue and the H^N of residue as an open triangle. NOEs between any atom of the side chain of residue and the H^N of residue are shown as a black circle.</p

¹⁵N Relaxation Data.

<p>Backbone 60.25 MHz ¹⁵N relaxation rates and ¹H-¹⁵N heteronuclear NOEs of 6 M urea-denatured TC5b acquired with 25 mM sodium phosphate buffer, pH 3.5, and at 298 K. (<b>A</b>) <i>R</i>₁ relaxation rates; (<b>B</b>) <i>R</i>₂ relaxation times; (<b>C</b>) ¹H-¹⁵N heteronuclear NOEs; (<b>D</b>) R₂/R₁ ratios.</p