Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution.

We propose a hypothesis that explains two apparently contradicting observations for the heterogeneity of the unfolded proteins. First, the line confocal method of the single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy revealed that the unfolded proteins possess broad peaks in the FRET efficiency plot, implying the significant heterogeneity that lasts longer than milliseconds. Second, the fluorescence correlation method demonstrated that the unfolded proteins fluctuate in the time scale shorter than 100 ns. To formulate the hypothesis, we first summarize the recent consensus for the structure and dynamics of the unfolded proteins. We next discuss the conventional method of the sm-FRET spectroscopy and its limitations for the analysis of the unfolded proteins, followed by the advantages of the line confocal method that revealed the heterogeneity. Finally, we propose that the structural heterogeneity formed by the local clustering of hydrophobic residues modulates the distribution of the long-range distance between the labeled chromophores, resulting in the broadening of the peak in the FRET efficiency plot. A clustering of hydrophobic residues around the chromophore might further contribute to the broadening. The proposed clusters are important for the understanding of protein folding mechanism.