Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain.

The N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) state and a relatively compact unfolded (U(exch)) state under nondenaturing conditions. Selectively labeled samples of the domain have been analyzed by NOESY NMR experiments to probe residual hydrophobic clustering in the U(exch) state. The labeling strategy included selective protonation of aromatic rings or delta-methyl groups on Ile and Leu residues in a highly deuterated background. Combined with long mixing times, the methods permitted observation of significant numbers of long-range interactions between hydrophobic side chains, providing evidence for multiple conformers involving non-native hydrophobic clusters around the Trp 36 indole. Comparison of these data with previously reported HN-HN NOEs yields structural insight into the diversity of structures within the U(exch) ensemble in the drkN SH3 domain. Many of the HN-HN NOEs are consistent with models containing compact residual nativelike secondary structure and greater exposure of the Trp 36 indole to solvent, similar to kinetic intermediates formed in the hierarchic condensation model of folding. However, the methyl and aromatic NOE data better fit conformations with non-native burial of the Trp indole surrounded by hydrophobic groups and more loosely formed beta-structure; these structural characteristics are more consistent with those of kinetic intermediates formed during the hydrophobic collapse mechanism of folding. This suite of NOE data provides a more complete picture of the structures that span the U(exch) state ensemble, from conformers with non-native structure but long-range contacts to those that are highly nativelike. Together, the results are also consistent with the folding funnel view involving multiple folding pathways for this molecule.