Multiple native states reveal persistent ruggedness of an RNA folding landscape

According to the 'thermodynamic hypothesis', the sequence of a biological macromolecule defines its folded, active (or 'native') structure as a global energy minimum in the folding landscape(1,2). However, the enormous complexity of folding landscapes of large macromolecules raises the question of whether there is in fact a unique global minimum corresponding to a unique native conformation or whether there are deep local minima corresponding to alternative active conformations(3). The folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation(4,5). Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert(6-8). Here we demonstrate, using single-molecule experiments, that an RNA enzyme folds into multiple distinct native states that interconvert on a timescale much longer than that of catalysis. These data demonstrate that severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.