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

Abstract

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 pK a 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.

Structural and spectroscopic characterization of photoactive yellow protein and photoswitchable fluorescent protein constructs containing heavy atoms

“…The absorption spectra of the PYP constructs are shown in Figure 4A with peak values listed in Table 3. Bromination of the pCA chromophore within PYP causes a small blue shift of 1 nm, similar to the observed blue shift for other halogenated chromophores in PYP [25] and other fluorescent proteins [30,31]. Franck-Condon excitation is purely an electronic process that reflects the electronic properties of the chromophore and the effect of the environment at the instantaneous moment of photon absorption before any atomic rearrangements.…”

“…Synthesis of the 3-bromo-pCA (3-Br-pCA) chromophore derivative followed by functionalization to the corresponding acid anhydride and linkage to the thiol of C69 yielded a PYP construct with the modified chromophore (PYP 3-Br-pCA, see Materials and Methods). Similar approaches in our lab and others have produced PYP variants with a less electron-rich substituent on the phenolate ring of pCA such as a hydroxy group [21][22][23], methoxy group [21,24], and chlorine atom [25]. Note that substituents have also been placed at the α position of the carboxylic acid of pCA (C3 in Fig.…”

“…Structural analysis of all PYP and rsEGFP2 constructs discussed above is important to determine the extent of perturbation caused by the heavy atoms and to provide starting coordinates for model building and data analysis in UED and TR-SFX experiments. The heavy-atom-containing PYP constructs were readily crystallized following similar protocols as previously described [25], and the crystals diffracted to atomic resolution (between 0.85 and 0.97 Å resolution, Table 1). The tertiary structures of all constructs align well with that of the wild-type protein (1NWZ, overall RMSD of less than 0.7 Å for the α-carbons, Table 1), with only slight deviations appearing in loop regions far from the chromophore, suggesting that the heavy atom incorporation does not alter the overall fold of the protein ( Fig.…”

Visualization of H atoms in the X‐ray crystal structure of photoactive yellow protein: Does it contain low‐barrier hydrogen bonds?

“…17 The proposal 6 that the HC4 E46 HB is a LBHB has led to many new experimental and theoretical studies. [18][19][20][21][22][23][24][25] Nearly, all of these studies (with single exception 22 ) have consistently showed that the HC4 E46 HB is not a LBHB. One study attempted to reconcile disagreements with new theoretical approaches, 22 but the validity of the new approaches has been questioned.…”

“…The proposal that the HC4E46 HB is a LBHB has led to many new experimental and theoretical studies . Nearly, all of these studies (with single exception) have consistently showed that the HC4E46 HB is not a LBHB.…”