Determination of an Ensemble of Structures Representing the Denatured State of the Bovine Acyl-Coenzyme A Binding Protein

The denatured state of a protein contains important information about the determinants of the
folding process. By combining site-directed spin-labeling NMR experiments and restrained computer
simulations, we have determined ensembles of conformations that represent the denatured state of the
bovine acyl-coenzyme A binding protein (ACBP) at three different concentrations of guanidine hydrochloride.
As the experimentally determined distance information corresponds to weighted averages over a broad
ensemble of structures, we applied the experimental restraints to a system of noninteracting replicas of
the protein by using a Monte Carlo sampling scheme. This procedure permits us to sample ensembles of
conformations that are compatible with the experimental data and thus to obtain information regarding the
distribution of structures in the denatured state. Our results show that the denatured state of ACBP is
highly heterogeneous. The high sensitivity of the computational method that we present, however, enabled
us to identify long-range interactions between two regions, located near the N- and C-termini, that include
both native and non-native elements. The preferential formation of these contacts suggests that the
sequence-dependent patterns of helical propensity and hydrophobicity are important determinants of the
structure in the denatured state of ACBP.