Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography, NMR and other biophysical techniques. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to mus regime) of a predominantly ( approximately 95%) open form (represented by the apo crystal structure) and a minor (approximately 5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a <r(-6)> ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.