Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.