Recently, the funnel shape energy landscape theory has been successfully utilized to describe the folding (1–4) and binding (5) behavior in proteins. While in general the free energy landscape is depicted by a funnel-like shape, the details of the landscape surface of a folding funnel will be affected by changes in the surrounding environment. On the basis of Freire’s work in this issue of the Proceedings (6), we describe a shift in the energy landscape of a folding funnel, caused by a binding event. Shifts in energy landscapes portray shifts in the populations of the substates. The exciting paper by Freire (6) presents a structure-based statistical thermodynamic approach to predict the stabilization effects observed following the binding of a ligand to a receptor. Interestingly, the structure-based thermodynamic analysis indicates that the stabilization effect is not distributed uniformly throughout the enzyme. Instead, upon the binding of the inhibitor, the stability constants of individual residues exhibit changes in their magnitudes. Of particular interest is the finding made by Freire and his colleagues that upon binding, large changes are observed in residues which are far away from the binding sites. These results are consistent with experiments (7) showing changes in amide hydrogen exchange rates in lysozyme, when complexed with three different antibodies. Regardless of the sites at which the (D44. 1, D1. 3, and HyHEL-5) antibodies bound, the remote residues were found to be protected by the binding events. Additional, consistent evidence comes from crystal structures of mutants of HIV-1 protease, which show that changes far away from the active site affect the conformation at this region (8).

Furthermore, a major finding of Freire is the dual character of active sites. Freire has applied the algorithm to a number of proteins, showing that binding sites characteristically have regions of high and low structural stability, with a significant fraction of their residues being flexible. While the less stable (more flexible) region allows ‘‘opening’’of the active site, to allow the ligand to enter, the stable region contributes to the specificity and affinity of the binding. This dual character implies that there are minor changes between the bound and unbound conformations at the active site. Hence, the algorithm developed by Freire (6) addresses cases that do not illustrate large scale motions, typically included in Koshland’s classical induced fit model (9). The availability of a predictive scheme, which can predict changes in stability of residues that are distant from the binding sites, and its application to studies of the effects of binding on remote regions are important contributions to our understanding of protein folding and binding.