Hidden complexity in the isomerization dynamics of Holliday junctions.

A plausible consequence of the rugged folding energy landscapes inherent to biomolecules is that there may be more than one functionally competent folded state. Indeed, molecule-to-molecule variations in the folding dynamics of enzymes and ribozymes have recently been identified in single-molecule experiments, but without systematic quantification or an understanding of their structural origin. Here, using concepts from glass physics and complementary clustering analysis, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time (T(obs) ≈ 40 s). We show that the ergodicity of Holliday junction dynamics is effectively broken and that their conformational space is partitioned into a folding network of kinetically disconnected clusters. Theory suggests that the persistent heterogeneity of Holliday junction dynamics is a consequence of internal multiloops with varying sizes and flexibilities frozen by Mg(2+) ions. An annealing experiment using Mg(2+) pulses lends support to this idea by explicitly showing that interconversions between trajectories with different patterns can be induced.