Perturbation of Short Hydrogen Bonds in Photoactive Yellow Protein via Noncanonical Amino Acid Incorporation

Photoactive yellow protein (PYP)
is a small photoreceptor protein
that has two unusually short hydrogen bonds between the deprotonated p-coumaric acid chromophore and two amino acids, a tyrosine
and a glutamic acid. This has led to considerable debate as to whether
the glutamic acid-chromophore hydrogen bond is a low barrier hydrogen
bond, with conflicting results in the literature. We have modified
the pKa of the tyrosine by amber suppression
and of the chromophore by chemical substitution. X-ray crystal structures
of these modified proteins are nearly identical to the wild-type protein,
so the heavy atom distance between proton donor and acceptor is maintained,
even though these modifications change the relative proton affinity
between donor and acceptor. Despite a considerable change in relative
proton affinity, the NMR chemical shifts of the hydrogen-bonded protons
are only moderately affected. QM/MM calculations were used to explore
the protons’ potential energy surface and connect the calculated
proton position with empirically measured proton chemical shifts.
The results are inconsistent with a low barrier hydrogen bond but
in all cases are consistent with a localized proton, suggesting an
ionic hydrogen bond rather than a low barrier hydrogen bond.