Low-barrier hydrogen bond in photoactiveyellow proteinShigeo Yamaguchia,1, Hironari Kamikuboa,1, Kazuo Kuriharab, Ryota Kurokib, Nobuo Niimurac, Nobutaka Shimizud,Yoichi Yamazakia, and Mikio Kataokaa,b,2

aGraduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan; bJapan Atomic EnergyAgency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195, Japan; cFrontier Research Center for Applied Atomic Sciences, Ibaraki University, 4-12-1Nakanarusawa, Hitachi, Ibaraki 316-8511, Japan; and dJapan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo, 679-5198, Japan

Communicated by Yasuyuki Yamada, Nara Institute of Science and Technology, Nara, Japan, November 23, 2008 (received for review September 10, 2008)

Low-barrier hydrogen bonds (LBHBs) have been proposed to playroles in protein functions, including enzymatic catalysis and protontransfer. Transient formation of LBHBs is expected to stabilizespecific reaction intermediates. However, based on experimentalresults and theoretical considerations, arguments against the im-portance of LBHB in proteins have been raised. The discrepancy iscaused by the absence of direct identification of the hydrogenatom position. Here, we show by high-resolution neutron crystal-lography of photoactive yellow protein (PYP) that a LBHB exists ina protein, even in the ground state. We identified �87% (819/942)of the hydrogen positions in PYP and demonstrated that thehydrogen bond between the chromophore and E46 is a LBHB. ThisLBHB stabilizes an isolated electric charge buried in the hydropho-bic environment of the protein interior. We propose that in theexcited state the fast relaxation of the LBHB into a normal hydro-gen bond is the trigger for photo-signal propagation to the proteinmoiety. These results give insights into the novel roles of LBHBsand the mechanism of the formation of LBHBs.

neutron crystallography � photoreaction � proton translocation �short hydrogen bond

The 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 ofcircumstantial evidence support the existence of LBHBs, nega-tive results have also been published (3–5). This discrepancy iscaused by the absence of direct demonstration of LBHBs inproteins. In general, hydrogen bonds in proteins are identified bythe distance between a donor and an acceptor within the crystalstructure. Because of its abnormally short bond length, a LBHBis accompanied by a quasi-covalent bond feature, whereas anordinary hydrogen bond can be depicted as an electrostaticinteraction between a donor–proton dipole and a dipole (or amonopole) on an acceptor atom (6–8). In LBHBs, the proton isshared by the donor and acceptor atoms, resulting in thedistribution of the hydrogen between the two (6). Therefore, toidentify a LBHB, it is essential to determine the position of thehydrogen atom and those of the donor and acceptor atoms.Recently, it was shown that a light sensor protein, photoactiveyellow 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 notbe 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 thepurple phototropic bacterium, Halorhodospira halophila (12).The chromophore of PYP, p-coumaric acid (pCA), is buried ina hydrophobic pocket. Absorption of a photon triggers theisomerization of the chromophore and the subsequent thermalreaction cycle (13, 14). The hydrogen-bonding network near thechromophore is modulated during the thermal reaction, result-ing in proton transfers within the network that are associatedwith large conformational changes (15–18). Two SHBs are

formed between pCA and E46 and between pCA and Y42. TheSHBs are OOH���O hydrogen bonds, with the phenolic oxygenof the chromophore located 2.51 Šfrom the phenolic oxygen ofY42 and 2.58 Šfrom the carboxylic oxygen of E46 (9). Theproton transfer occurs between E46 and the chromophoreduring the thermal reaction (15, 16). Therefore, the SHBs areessential for the photoreaction and stability of PYP.

In this article, to reveal the properties of the SHBs in PYP, weperformed high-resolution neutron crystallographic analysiscombined with high-resolution X-ray crystallography. The largecrystals, prepared by using the crystallization phase diagrammethod (19), diffracted neutron and X-ray up to 1.5- and 1.25-Åresolution, respectively, at room temperature. Using the jointmethod of the X-ray and neutron refinements, clear nucleardensity maps of 87% of hydrogen and deuterium atoms in thewhole protein were observed. From the nuclear density maps, wesucceeded in identifying the deuterium atoms involved in theSHBs and determined that the SHB between pCA and E46 is aLBHB, whereas the SHB between pCA and Y42 is not a LBHBbut should be termed as the short ionic hydrogen bond (SIHB).We also revealed that R52, which is believed to be protonated tobe a counter ion of pCA, is deprotonated. Finally, based on theobservation, we discuss roles of the LBHB in the protein.

Results and DiscussionObservation of the Hydrogen Atoms of PYP. To identify the positionsof hydrogen and deuterium atoms precisely, a joint method ofneutron and X-ray crystallography was applied to neutrondiffraction data at 1.5-Å resolution and X-ray diffraction data at1.25-Å resolution, and the positions of 87% of hydrogen anddeuterium atoms in PYP were determined (Fig. 1A). Fig. 1Bshows the hydrogen-bonding network, including all hydrogen/deuterium atoms around the chromophore. The details of thehydrogen bonds are shown in Fig. 1 C–F, onto which thedistributions of the heavy atom electron density and the deute-rium/hydrogen atom nuclear density are superimposed. Allhydrogen/deuterium atoms responsible for hydrogen bonds areclearly observed. The distances from the phenolic oxygen of thechromophore to the phenolic oxygen of Y42 and to the carbox-ylic oxygen of E46 are 2.52 and 2.56 Å, respectively (Fig. 1C).These distances are altered by a few 0.01 Å compared with theprevious X-ray crystal structure (9). This subtle difference wasalso observed by the previous neutron study (10), suggesting the

Author contributions: M.K. designed research; S.Y., H.K., K.K., R.K., N.N., N.S., and Y.Y.performed research; S.Y. and H.K. analyzed data; and H.K. and M.K. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 2ZOH and 2ZOI).

1S.Y. and H.K. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: kataoka@ms.naist.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811882106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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effects of the H–D exchange. The hydrogen-bonding networkacross E46 and Y42 is spread over the molecule and reaches theN-terminal region (Fig. 1D) and the loop connecting �4 and �5through R52 (Fig. 1E). These regions exhibit substantial con-formational changes during the photo-reaction process, ulti-mately leading to signaling (18, 20). Another important findingis that R52 is deprotonated (Fig. 1E), although R52 is believedto be protonated to serve as the counterion of the negatively-charged chromophore (21).

Hydrogen Bonds in PYP. According to the distances between donorand acceptor atoms, PYP contains 115 hydrogen bonds, amongwhich 103 hydrogen (or deuterium) atoms exhibited clear nu-clear densities. Fig. 2A shows the correlation of donor-H(D) and

H(D)-acceptor bond lengths with the donor–acceptor distancesand also the histogram of hydrogen-bond lengths. Among these,2 SHBs exhibit distances significantly shorter than those of theother hydrogen bonds. Except for the SHB of pCA-E46, thedonor-H(D) distances are nearly constant. Average distances are0.95 Å for O-H(D) and 0.98 Å for N-H(D), and the H(D)-acceptor distances range from 1.8 to 2.5 Å with an averagedistance of 2.07 Å. Hydrogen atoms involved in these hydrogenbonds were replaced by deuterium atoms except for thoselocated in some of the �-strands. The average distances of theNH and ND bonds were, however, indistinguishable at thepresent resolution. The nuclear density maps of the deuteriumatoms involved in the two SHBs can be clearly observed (Fig. 2B and C). The interatomic distances involved in these hydrogen

Fig. 1. Structure of PYP including hydrogen atom positions. (A) Stereoview of the whole molecule including the determined hydrogen and deuterium atoms.(B) Stereoview of the hydrogen bonding network around the chromophore. (C–F) Stereoviews of the representative hydrogen bonds shown in B. The FO � FC

difference nuclear density map omitting the hydrogen/deuterium atoms, superimposed on the structural model is shown. The blue mesh (contoured at 5.5 �)represents the positive nuclear density of the deuterium atoms, and the red mesh (contoured at �5.5 �) represents the negative nuclear density of the hydrogenatoms. The yellow mesh (contoured at 4.0 �) shows the 2FO � FC electron density map calculated from X-ray crystallographic analysis, which was used for thedetermination of the heavy atom positions.

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bonds are listed in Fig. 2 B and C. In the SHB between pCA andE46, the distances between the phenolic oxygen of pCA and Dand between the carboxylic oxygen of E46 and D are 1.37 and1.21 Å, respectively. Both bond lengths are substantially longerthan the average lengths of OOH (OOD) covalent bonds (0.95Å), indicating that the deuterium atom is not covalently boundto either of the two oxygen atoms. However, these bond lengthsare much shorter than the average H(D)-acceptor bond length(2.07 Å) (see Fig. 2 A). These values indicate that the deuteriumatoms must be shared by both oxygen atoms. We can concludethat the SHB between the chromophore and E46 is a LBHB. Incontrast, the position of the deuterium atom between Y42 and

pCA is shifted toward the phenolic oxygen atom of Y42. Theinteratomic distance between the phenolic oxygen of Y42 and Dis 0.96 Å, close to the average covalent OOH bond length, 0.95Å. Therefore, the deuterium atom is covalently bound to thephenolic oxygen atom of Y42. The interatomic distance betweenthe phenolic oxygen of pCA and D is 1.65 Å, which is shorter thanthe average distance between H(D) and acceptor atom (O), 2.07Å. The SHB between pCA and Y42 is not a LBHB, but rathera SIHB. Thus, a SHB is not always a LBHB.

Role of LBHB in PYP. It has been proposed that pCA takes ananionic form (15, 16). In general, an isolated charge buried in aprotein’s interior will destabilize the protein structure; to beelectrically neutralized, it must be coupled with a counterion.The counterion against pCA’s negative charge is thought to bethe cationic form of R52 at a distance of 6.34 Å from the phenolicoxygen of pCA (21) (Fig. 3A). However, as shown in Fig. 1E, R52takes an electrically neutral form in PYP, indicating that theisolated negative charge must be stabilized by another mecha-nism. One possible explanation can be retrieved by the strongbond strength of LBHB. The bond strength of LBHB is 12–24kcal/mol (6), which is extremely stronger than that of an ordinaryionic hydrogen bond (a few kcal/mol). Therefore, the energeticgain by the LBHB compensates the energetic disadvantage of theisolated charge buried in the protein interior. We also proposethat the isolated negative charge is stabilized partly because ofthe delocalization of the negative charge within the LBHB-conjugated system (Fig. 3B). This proposal is based on a valencebond representation of LBHB (5, 8, 22). LBHB connecting�-conjugated systems are often observed in homoconjugatedbetaine complexes (8, 22). Although the stretching frequency ofasymmetric CO in a carboxyl group usually appears at �1,780cm�1, the stretching frequency in homoconjugated betainecomplexes is shifted toward the lower frequency, ranging from1,720 to 1,740 cm�1. The asymmetric CO stretching vibration of

Fig. 2. Hydrogen bonds in PYP. (A) Correlation of donor-H(D) and H(D)-acceptor bond lengths with the donor–acceptor distances and a histogram ofhydrogen bond lengths. The open and closed triangles show the donor-H(D)and the H(D)-acceptor distances, respectively. The SHBs are shown as a redcircle (pCA-E46) and a blue circle (pCA-Y42). The solid lines represent thecalculated donor-H(D) distances, assuming bent angles of 120° and 180°,respectively. (B and C) The nuclear and electron density maps with the struc-ture models of the two SHBs, pCA-E46 (B) and pCA-Y42 (C). The blue meshrepresents the positive nuclear density of the deuterium atoms, contoured at80% of the maximum peak height of the FO � FC difference maps omittingeach deuterium atom involved in the hydrogen bond; contour levels of 80%of the maximum peak height correspond to 8.26 � for pCA-E46 and 6.86 � orpCA-Y42. The yellow mesh (contoured at 4.1 �) and the red mesh (contouredat �5.3 �) show the 2FO � FC electron density maps of the heavy atoms and theFO � FC difference nuclear density map omitting the hydrogen atoms,respectively.

Fig. 3. The mechanism of the stabilization of the isolated negative charge inthe vicinity of the chromophore in a hydrophobic environment of PYP. (A) Theneutralization by a counter ion, which is believed so far. We ruled out thismechanism by the finding that R52 is not protonated (Fig. 1E). (B) Strong bondstrength of LBHB and charge delocalization caused by the quasi-covalentbond of LBHB stabilizes the energetic disadvantage of the isolated negativecharge buried in the protein interior.

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the carboxyl group of E46 was observed at 1,739 cm�1 (15, 16).The good agreement in the CO stretching frequency supportsour proposal in Fig. 3B that the LBHB combines the two�-conjugated systems of E46 and pCA. As a consequence, anegative charge is delocalized over the conjugated system com-prised of the two �-conjugate bond groups.

The existence of the LBHB between pCA and E46 indicatesthat the proton affinities of pCA and E46 are close to each otherin the protein interior (6, 22), even though in solution the pKavalues of pCA and the carboxyl group of glutamic acid are 8.8(23) and 4.25, respectively. The pKa increase of E46 is caused bythe transfer of glutamic acid into the hydrophobic environmentof the protein interior, and the pKa decrease of pCA is partlycaused by the stabilization of the deprotonated resonance formof pCA, with the aid of the hydrogen bond (NOH���O) betweenthe amide proton of C69 and the carbonyl oxygen of pCA (24)(see Fig. 1F). As a consequence, in the dark state, pCA and E46show similar pKa values in the protein interior. In the excitedstate, an instantaneous change in dipole moment brings about acharge translocation from the phenol ring to the ethylene chainof the chromophore (25). This charge translocation leads toalteration of the proton affinity of the phenolic oxygen, resultingin relaxation of the LBHB into an ordinary ionic hydrogen bond;the shared proton is transferred to E46 during the lifetime of theexcited state (�1 ps). Ultra-fast infrared spectroscopy hasrevealed that the CO stretching mode of the carboxyl group ofE46 is altered during formation of the excited state (26). In theground state, the quasi-covalent bond of the LBHB can beexpected to sterically restrain the phenol ring moiety of thechromophore at the specific position. Once the LBHB is disruptedin the excited state, the phenol ring moiety of the chromophore isliberated and promotes the subsequent molecular events, specifi-cally, the fast isomerization of the chromophore.

LBHBs have been proposed to be responsible for enzymaticcatalysis, in particular, in the transition state of the catalyticcenters of serine proteases (1, 2). However, the transient for-mation of LBHBs has not been confirmed to our knowledge untilthis study. Our data clearly show that LBHB can be formed ina protein even in the ground state. We conclude that the pKamatching between donor and acceptor atoms is the prerequisitefor the formation of LBHB, which provides an insight into thetransient formation of LBHB. The present observation of aLBHB involved in PYP reveals roles of LBHB in the biophysicalaspects of protein structure and function, i.e., stabilization of anisolated charge in the protein interior and mediation of fastproton transfer in the excited state. Recently, spectroscopicstudy of a GFP variant proposed that fast excited-state protontransfer near the chromophore is facilitated by a LBHB (27),suggesting that the properties of LBHB proposed here are widelyused. The extensive experimental and theoretical studies of theLBHB of PYP will establish additional roles for hydrogen bondsand help develop our understanding of the molecular mecha-nisms operating in a wide range of proteins.

MethodsCrystallization of PYP for Neutron and X-Ray Diffraction Experiments. Wild-typePYP was overexpressed by using the pET system in Escherichia coli BL21(DE3)(Novagen) and reconstituted with pCA anhydride in 4 M urea buffer (28). Theproteins were purified by using DEAE Sepharose CL6B (Amersham Biosciences)column chromatography several times until the optical purity index (absor-bance 277 nm/absorbance �max) became �0.44. Crystals of PYP were preparedby the hanging-drop vapor diffusion method in conjunction with the micro-seeding method (a useful means of regulating the number of seeds in a drop)under the supersaturation conditions (19). The crystallization drop solutionwas adjusted to 24 mg�mL�1 PYP, 2.2 M ammonium sulfate, and 1 M sodiumchloride with 20 mM sodium phosphate buffer. The reservoir solution was 2.5M ammonium sulfate and 1.1–1.2 M sodium chloride. These crystallizationbuffers were made with 99.9% heavy water (Aldrich). The temperature andpD of these solutions were maintained at 293 K and 9.0, respectively.

X-Ray Data Collection, Processing, and Refinement. The X-ray diffractionexperiments were performed at room temperature by using the BL41XU beamline installed at SPring-8, Hyogo, Japan. The crystal (2.37 � 0.70 � 0.66 mm3)was sealed in a quartz capillary. The diffraction data were recorded with aQuantum 315 CCD detector (ADSC) over a rotation of 180° with an oscillationstep of 1.0°. The wavelength was set to 0.45 Å. The camera distance andexposure time for each image were 300 mm and 1 s, respectively. The diffrac-tion data up to 1.25 Å were processed with XDS (29) and SCALA (30). The Rmerge

of each diffraction image was not altered during the diffraction experiment,suggesting that the radiation damage was negligibly small.

The initial phases were determined by using the molecular replacementprogram AMoRe (31); the previously reported structural model (Protein DataBank ID code 2PHY; ref. 21) was used as the initial model. The refinementprograms CNS (32) and SHELXL (33) were used to refine the structural model,and Coot (34) was used to build the model. Finally, the anisotropic B factorswere refined. During the analysis, several residues were found to occupy morethan one conformation; these multiple conformations were subsequentlybuilt and refined. The final values of R and Rfree were 11.2% and 14.9%,respectively (Table S1). Model quality was assessed by PROCHECK (35) imple-mented in CCP4. All residues displayed � and � values in the favored orallowed regions of the Ramachandran plot. The detailed statistics of datacollection are shown in Table S1.

Neutron Data Collection and Processing. The neutron diffraction experimentswere carried out at room temperature by using a neutron single crystaldiffractometer (BIX-4) installed at the JRR-3 reactor, Japan Atomic EnergyAgency (36). The crystal (2.89 � 0.85 � 0.79 mm3) was sealed in a quartzcapillary. The neutron wavelength was set to 2.6 Å. A sufficient amount ofreservoir solution was added to the bottom of the capillary to prevent thecrystal from drying. The data were collected according to the step-scanmethod, with an oscillation step of 0.3°. The collection time for each frame was4 h. To improve completeness, after collecting 118 frames, the capillary wasrotated by 90° on the plane perpendicular to the incident neutron beam, andan additional 143 frames of the � step-scan were recorded. The net time requiredtocollect thetotalof261frameswas45days.Thedataset fromthePYPcrystalwasprocessed up to 1.5-Å resolution by using the programs DENZO and SCALEPACK(37), both of which were suitably modified for the processing of neutron data.The detailed statistics of data collection are shown in Table S1.

Refinement of the Hydrogen/Deuterium Atom Positions. Molecular refinementfor the neutron crystallographic analysis was initiated by using the structuralmodel determined by the X-ray crystallographic analysis, with the watermolecules removed. The crystals used for the X-ray and neutron diffractionexperiments were grown under the same crystallization conditions, suggest-ing that the observed molecular structures were potentially identical to eachother (Table S1). Structure determination was performed by using the refine-ment programs X-PLOR (38) and CNS (32), in which the topology and param-eter files had been specially modified for neutron crystallography. The pro-grams Coot (34) and XtalView (39) were used to build the model. Although thepositions of the heavy atoms determined by the X-ray crystallographic analysiswere fixed during the refinement procedure, the positions of the hydrogenatoms, deuterium atoms, and water molecules were refined with rigid body,annealing refinement and energy minimization procedures using the neutrondiffraction data. The joint method of neutron and X-ray crystallographyimproved nuclear density map of hydrogen and deuterium atoms, whencompared with the ordinary neutron crystallography (see SI Text and Fig. S1)Although 120 water molecules are visible in the X-ray crystallographic anal-ysis, only 73 of the water molecules can be observed in the neutron crystal-lographic analysis. However, the neutron crystallographic analysis revealedthree types of water molecules with different types of rotational freedom (40,41); the number of molecules of the triangular type, short ellipsoidal sticktype, and spherical type are 25, 10, and 38, respectively (Table S1).

Preparation of the Figures. PyMOL was used in the preparation of the nuclearand electron density maps superimposed on the ball-and-stick model in Figs.1 and 2 and Fig. S1 (42).

ACKNOWLEDGMENTS. We thank Dr. Yuki Ohnishi (Ibaraki University) for helpin the beginning stage of crystallization. This work was supported in part byfrom the Ministry of Education, Culture, Sports, Science, and Technology ofJapan Grants-in-Aid for Scientific Research 15076208 and 20050020 (to M.K.).The neutron diffraction experiments were performed under the approval ofthe common-use facility program of the Japan Atomic Energy Agency (pro-posal 2007A-A08). The X-ray diffraction experiments were performed underthe approval of the Japan Synchrotron Radiation Research Institute ProgramAdvisory Committee (proposal 2006B1765).

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