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...

Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and ðNH 4 Þ 2 HPO 4 . The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the ΔH of the oligomer dissociation to monomers was estimated to be about −20 kcal∕mol per protomer unit, where Met-heme coordination appears to contribute largely to ΔH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.dimer | trimer | protein polymer

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90°out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/ relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.time-resolved X-ray diffraction | photoreceptor | light sensor

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5°C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.The active site of an ion pump must communicate alternately with the two opposite sides of the membrane. Change of the protein conformation, linked to this switch, is therefore expected to be an essential step in the reaction cycle. In the light-driven proton pump bacteriorhodopsin (bR), the switch must occur after proton transfer toward the extracellular side but before proton transfer from the cytoplasmic side-i.e., while the proton binding site, the retinal Schiff base, is unprotonated. Thus, the switch reaction is expected to take place during the lifetime of the M intermediate in what was termed the Ml to M2 reaction (1-5). Structural changes are indeed revealed by neutron, x-ray, and electron diffraction when photostationary states are created where the M intermediate accumulates (6-9). We have shown that this structural change is closely related to the deprotonation of Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C) it is open to the cytoplasmic side (10). Thus, the proton transport mechanism is elegantly explained by the alternating protein conformation model (11, 12). Light energy is utilized to cause the initial proton transfer that triggers the structural transition from conformation E to conformation C, and that results in the change of access of the retinal Schiff base as well as further changes in proton affinity (pK) of donor and acceptor groups so as to drive proton transfers at strategic locations in the protein.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

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.

SUMMARYAlthough the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment 'IZUMO1 putative functional fragment (IZUMO1 PFF )'. Surprisingly, IZUMO1 PPF still maintains binding ability on the egg surface of Cd9 −/− eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1 PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1 PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1 PFF-core .

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The light-induced global conformational change of photoactive yellow protein was directly observed by small-angle X-ray scattering (SAXS). The N-terminal 6, 15, or 23 amino acid residues were enzymatically truncated (T6, T15, or T23, respectively), and their near-UV intermediates were accumulated under continuous illumination for SAXS measurements. The Kratky plot demonstrated that illumination induced partial loss of globularity. The change in globularity was marked in T6 but very small in T15 and T23, suggesting that structural change in positions 7-15 mainly reduces the globularity. The radius of gyration (R(g)) estimated by Guinier plot was increased by 1.1 A for T6 and 0.7 A for T15 and T23 upon illumination. As T23 lacks most of the N-terminal loop, structural change in the main part composed of the PAS core, helical connector, and beta-scaffold caused an increase of R(g) by 0.7 A. The structural change of positions 7-15 caused an additional increase by 0.4 A. The decrease of R(g) upon truncation of positions 7-15 for dark state was 0.3 A, while that for the intermediate was 0.7 A, suggesting that this region moves outward on formation of the intermediate. These results indicate that a light-induced structural change of PYP takes place in the main part and N-terminal 15 amino acid residues. The former induces only dimensional increase, but the latter results in additional change in shape.