A Dynamic Equilibrium of Three Hydrogen-Bond Conformers Explains the NMR Spectrum of the Active Site of Photoactive Yellow Protein

A theoretical
study on the NMR shifts of the hydrogen bond network
around the chromophore, para-coumaric acid (pCA),
of photoactive yellow protein (PYP) is presented. Previous discrepancies
between theoretical and experimental studies are resolved by our findings
of a previously unknown rapid conformational exchange near the active
site of PYP. This exchange caused by the rotation of Thr50 takes place
in the ground state of PYP’s active site and results in three
effectively energetically equal conformations characterized by the
formation of new hydrogen bonds, all of which contribute to the overall
NMR signals of the investigated protons. In light of these findings,
we are able to successfully explain the experimental results and provide
valuable insight into the behavior of PYP in solution. We further
investigated related PYP mutants (T50V, E46Q, and Y42F), and found
the same conformational exchange in E46Q and Y42F to be responsible
for the experimentally observed NMR and UV/vis spectra.