Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is caused by the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified Ϸ87% (819/942) of the hydrogen positions in PYP and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs and the mechanism of the formation of LBHBs.neutron crystallography ͉ photoreaction ͉ proton translocation ͉ short hydrogen bond T he idea that the formation of low-barrier hydrogen bonds (LBHBs) plays an essential role in enzyme catalysis was proposed in the early 1990s (1, 2). Although several lines of circumstantial evidence support the existence of LBHBs, negative results have also been published (3-5). This discrepancy is caused by the absence of direct demonstration of LBHBs in proteins. In general, hydrogen bonds in proteins are identified by the distance between a donor and an acceptor within the crystal structure. Because of its abnormally short bond length, a LBHB is accompanied by a quasi-covalent bond feature, whereas an ordinary hydrogen bond can be depicted as an electrostatic interaction between a donor-proton dipole and a dipole (or a monopole) on an acceptor atom (6-8). In LBHBs, the proton is shared by the donor and acceptor atoms, resulting in the distribution of the hydrogen between the two (6). Therefore, to identify a LBHB, it is essential to determine the position of the hydrogen atom and those of the donor and acceptor atoms. Recently, it was shown that a light sensor protein, photoactive yellow protein (PYP), contains 2 short hydrogen bonds (SHBs) adjacent to the reaction center, even in the ground state (9, 10). The hydrogen atoms involved in the SHBs, however, could not be observed either by X-ray crystallography at atomic resolution (9, 11) or neutron crystallography at 2.5-Å resolution (10).PYP is a putative photoreceptor for negative phototaxis of the purple phototropic bacterium, Halorhodospira halophila (12). The chromophore of PYP, p-coumaric acid (pCA), is buried in a hydrophobic pocket. Absorption of a photon triggers the isomerization of the chromophore and the subsequent thermal reaction cycle (13,14). The hydrogen-bonding network near the chromophore is modulated during the thermal reaction, result...

Photoactive yellow protein (PYP) is photoconverted to its putative active form (PYP(M)) with global conformational change(s). The changes in the secondary structure were studied by far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy using PYP, which lacks N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively). Irradiation of truncated PYPs induced the loss of the CD signal, where the maximal difference was located at 222 nm. The reduction of the CD signal was significantly larger than the calculated CD of the N-terminal helices, indicating that it is mainly accounted for by the unfolding and/or structural change of the helices located outside the N-terminal region. The difference FTIR spectra between dark and photosteady states recorded using the solution samples demonstrated that large absorbance changes in the amide mode of the beta-sheet were reduced and downshifted by truncation. The structural change of the beta-sheet is therefore closely correlated with the N-terminal loop. NaCl decelerates the decay of intact PYP(M) and T6(M) at low concentrations (<500 mM) but accelerates decay at high concentrations (>1000 mM). For T15(M) and T23(M), NaCl accelerates their decay at >100 mM but never decelerates their decay, suggesting that the electrostatic interaction, which plays an important role for the recovery of PYP from PYP(M), is lost by removing positions 7-15. The electrostatic interaction between this region and the beta-scaffold is likely to promote the conformational change of PYP(M) for recovery of PYP.

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The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.

Fourier-transform infrared spectra were recorded at 170 K before and after irradiating the Asp85-->Asn mutant of bacteriorhodopsin. The difference spectrum exhibits protein bands such as those due to the perturbations of Asp96 and Asp115 and the N-H stretching vibration of tryptophan, characteristic of the L minus all-trans-bacteriorhodopsin spectrum of the wild-type protein. However, some vibrational bands of the peptide backbone and the chromophore are different from L and more characteristic of N of the wild-type protein. Remarkably, the shift observed for the vibrational band due to an internal water molecule upon L formation [Maeda, Sasaki, Shichida, and Yoshizawa (1992) Biochemistry 31, 462-467] is absent. These changes in the spectrum of the mutant could originate from the destruction of a hydrogen-bonding system consisting of Asp85, the water molecule, and the Schiff base, upon replacement of Asp85 with asparagine. These observations constitute direct evidence for the interaction of water with Asp85 at the time when it is protonated by the Schiff base.