  A plausible consequence of rugged energy landscapes of biomolecules is that
functionally competent folded states may not be unique, as is generally
assumed. Indeed, molecule-to-molecule variations in the dynamics of enzymes and
ribozymes under folding conditions have recently been identified in single
molecule experiments. However, systematic quantification and the structural
origin of the observed complex behavior remain elusive. Even for a relatively
simple case of isomerization dynamics in Holliday Junctions (HJs), molecular
heterogeneities persist over a long observation time (Tobs ~ 40 sec). Here,
using concepts in glass physics and complementary clustering analysis, we
provide a quantitative method to analyze the smFRET data probing the
isomerization in HJ dynamics. We show that ergodicity of HJ dynamics is
effectively broken; as a result, the conformational space of HJs is partitioned
into a folding network of kinetically disconnected clusters. While
isomerization dynamics in each cluster occurs rapidly as if the associated
conformational space is fully sampled, distinct patterns of time series
belonging to different clusters do not interconvert on Tobs. Theory suggests
that persistent heterogeneity of HJ dynamics is a consequence of internal
multiloops with varying sizes and flexibilities frozen by Mg2+ ions. An
annealing experiment using Mg2+ pulse that changes the Mg2+ cocentration from
high to low to high values lends support to this idea by explicitly showing
that interconversions can be driven among trajectories with different patterns.
