Abstract
Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.