Stretching vibrational frequencies and pKa differences in H-bond networks of protein environments.

The experimentally measured stretching vibrational frequencies of O-D [νO-D(donor)] and C=O [νC=O(donor)] H-bond donor groups can provide valuable information about the H-bonds in proteins. Here, using a quantum mechanical/molecular mechanical approach, the relationship between these vibrational frequencies and the difference in pKa values between H-bond donor and acceptor groups [ΔpKa(donor … acceptor)] in bacteriorhodopsin and photoactive yellow protein environments was investigated. The results show that νO-D(donor) is correlated with ΔpKa(donor … acceptor), regardless of the specific protein environment. νC=O(donor) is also correlated with ΔpKa(donor … acceptor), although the correlation is weak because the C=O bond does not have a proton. Importantly, the shifts in νO-D(donor) and νC=O(donor) are not caused by changes in pKa(donor) alone, but rather by changes in ΔpKa(donor … acceptor). Specifically, a decrease in ΔpKa(donor … acceptor) can lead to proton release from the H-bond donor group toward the acceptor group, resulting in shifts in the vibrational frequencies of the protein environment. These findings suggest that changes in the stretching vibrational frequencies, in particular νO-D(donor), can be used to monitor proton transfer in protein environments.

pH-Dependent Binding and Releasing Mechanism of Acetate in the Inner Water Cavity of Heliorhodopsin.

The high-resolution structure of heliorhodopsin crystallized at low pH reveals the presence of a planar triangle molecule, acetate, in the inner water cavity. Here, we investigate how the acetate molecule is stabilized at the counterion Glu107 moiety, using molecular dynamics (MD) simulations and a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations indicate that the density is best described as acetate among triangle acids, including nitric acid and bicarbonate. The calculated protonation state indicates that protonated acetate donates an H-bond to deprotonated Glu107 in the low-pH crystal structure. The observed red-shift of ∼30 nm in the absorption wavelength with pKa ≈ 4 is likely due to the His23/His80 protonation, rather than the Glu107 protonation. MD simulations also show that acetate can exist at the Glu107 moiety only when it is protonated. When ionized, acetate is released from the Glu107 moiety via Asn101 at the channel bottleneck and Arg91 on the intracellular protein surface. These observations could explain how acetate binds at low pH and releases at high pH.

Identification of intermediate conformations in the photocycle of the light-driven sodium-pumping rhodopsin KR2.

The light-driven rhodopsin KR2 transports Na+via the M- and O-states. However, the mechanisms by which the retinal regulates Na+ pumping is unknown, in part because KR2 adopts both pentamer and monomer forms in crystal structures and in part because these structures show differences in the protein conformation near the Schiff base, even when they are of the same intermediate state within the photocycle. A particular open question is the nature of the H-bond networks and protonation state in the active site, including Asp116. Here, we analyze the protonation state and the absorption wavelength for each crystal structure, using a quantum mechanical/molecular mechanical approach. In the pentamer ground state, the calculated absorption wavelength reproduces the experimentally measured absorption wavelength (530 nm). The analysis also shows that ionized Asp116 is stabilized by the H-bond donations of both Ser70 and a cluster of water molecules. The absorption wavelength of 400 nm in the M-state can be best reproduced when the two O atoms of Asp116 interact strongly with the Schiff base, as reported in one of the previous monomer ground state structures. The absorption wavelengths calculated for the two Na+-incorporated O-state structures are consistent with the measured absorption wavelength (∼600 nm), which suggests that two conformations represent the O-state. These results may provide a key to designing enhanced tools in optogenetics.

Ectopic expression of the human MutT-type Nudix hydrolase, hMTH1, confers enhanced tolerance to oxidative stress in arabidopsis.

Oxidized nucleotides produced by oxidative stress cause DNA mutations and the production of abnormal proteins. Thus, mammalian cells have developed multiple MutT-type Nudix hydrolases that exhibit pyrophosphohydrolase activity toward oxidized nucleotides in the cytosol, mitochondria and nucleus. On the other hand, AtNUDX1 is the only MutT-type Nudix hydrolase in the cytosol of Arabidopsis plants. To clarify the physiological significance of the defenses against oxidatively induced DNA damage in plant organelles, we analyzed the effects of the ectopic expression of the human MutT-type Nudix hydrolase, hMTH1, which was localized in the cytosol (cyt-hMTH1), chloroplasts (chl-hMTH1) and mitochondria (mit-hMTH1) of Arabidopsis cells, on tolerance to oxidative stress. Tolerance to oxidative stress caused by heating and paraquat (PQ) treatment was higher in the mit-hMTH1 and chl-hMTH1 plants than in the control and cyt-hMTH1 plants. The accumulation of H2O2 and the frequency of dead cells were lower in the mit-hMTH1 and chl-hMTH1 plants under stressful conditions. The poly(ADP-ribosyl)ation (PAR) reaction, which regulates repair systems for damaged DNA, was activated in the mit-hMTH1 and chl-hMTH1 plants under heat stress and PQ treatment. Furthermore, DNA fragmentation, which caused programmed cell death, was clearly suppressed in the mit-hMTH1 and chl-hMTH1 plants under heat stress. These results demonstrated that the ectopic expression of hMTH1 in the chloroplasts and mitochondria of Arabidopsis enhanced oxidative stress tolerance by activating the PAR reaction and suppressing programmed cell death.

Mechanism of Absorption Wavelength Shift Depending on the Protonation State of the Acrylate Group in Chlorophyll c.

Diatoms can use light in the blue-green region because they have chlorophyll c (Chlc) in light-harvesting antenna proteins, fucoxanthin and chlorophyll a/c-binding protein (FCP). Chlc has a protonatable acrylate group (-CH═CH-COOH/COO-) conjugated to the porphyrin ring. As the absorption wavelength of Chlc changes upon the protonation of the acrylate group, Chlc is a candidate component that is responsible for photoprotection in diatoms, which switches the FCP function between light-harvesting and energy-dissipation modes depending on the light intensity. Here, we investigate the mechanism by which the absorption wavelength of Chlc changes owing to the change in the protonation state of the acrylate group, using a quantum mechanical/molecular mechanical approach. The calculated absorption wavelength of the Soret band of protonated Chlc is ∼25 nm longer than that of deprotonated Chlc, which is due to the delocalization of the lowest (LUMO) and second lowest (LUMO+1) unoccupied molecular orbitals toward the acrylate group. These results suggest that in FCP, the decrease in pH on the lumenal side under high-light conditions leads to protonation of Chlc and thereby a red shift in the absorption wavelength.

Absorption wavelength along chromophore low-barrier hydrogen bonds.

In low-barrier hydrogen bonds (H-bonds), the pK a values for the H-bond donor and acceptor moieties are nearly equal, whereas the redox potential values depend on the H+ position. Spectroscopic details of low-barrier H-bonds remain unclear. Here, we report the absorption wavelength along low-barrier H-bonds in protein environments, using a quantum mechanical/molecular mechanical approach. Low-barrier H-bonds form between Glu46 and p-coumaric acid (pCA) in the intermediate pRCW state of photoactive yellow protein and between Asp116 and the retinal Schiff base in the intermediate M-state of the sodium-pumping rhodopsin KR2. The H+ displacement of only ∼0.4 Å, which does not easily occur without low-barrier H-bonds, is responsible for the ∼50 nm-shift in the absorption wavelength. This may be a basis of how photoreceptor proteins have evolved to proceed photocycles using abundant protons.

Proton transfer and conformational changes along the hydrogen bond network in heliorhodopsin.

Heliorhodopsin releases a proton from the Schiff base during the L-state to M-state transition but not toward the protein bulk surface. Here we investigate proton transfer and induced structural changes along the H-bond network in heliorhodopsin using a quantum mechanical/molecular mechanical approach and molecular dynamics simulations. Light-induced proton transfer could occur from the Schiff base toward Glu107, reorienting Ser76, followed by subsequent proton transfer toward His80. His80 protonation induces the reorientation of Trp246 on the extracellular surface, originating from the electrostatic interaction that propagates along the transmembrane H-bond network [His80…His23…H2O[H23/Q26]…Gln26…Trp246] over a distance of 15 Å. Furthermore, it induces structural fluctuation on the intracellular side in the H-bond network [His80…Asn16…Tyr92…Glu230…Arg104…Glu149], opening the inner cavity at the Tyr92 moiety. These may be a basis of how light-induced proton transfer causes conformational changes during the M-state to O-state transition.

Proton transfer pathway in anion channelrhodopsin-1.

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative.

Rhodopsins act as photoreceptors with their chromophore retinal (vitamin-A aldehyde) and they regulate light-dependent biological functions. Archaerhodopsin-3 (AR3) is an outward proton pump that has been widely utilized as a tool for optogenetics, a method for controlling cellular activity by light. To characterize the retinal binding cavity of AR3, we synthesized a dimethyl phenylated retinal derivative, (2E,4E,6E,8E)-9-(2,6-Dimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (DMP-retinal). QM/MM calculations suggested that DMP-retinal can be incorporated into the opsin of AR3 (archaeopsin-3, AO3). Thus, we introduced DMP-retinal into AO3 to obtain the non-natural holoprotein (AO3-DMP) and compared some molecular properties with those of AO3 with the natural A1-retinal (AO3-A1) or AR3. Light-induced pH change measurements revealed that AO3-DMP maintained slow outward proton pumping. Noteworthy, AO3-DMP had several significant changes in its molecular properties compared with AO3-A1 as follows; 1) spectroscopic measurements revealed that the absorption maximum was shifted from 556 to 508 nm and QM/MM calculations showed that the blue-shift was due to the significant increase in the HOMO-LUMO energy gap of the chromophore with the contribution of some residues around the chromophore, 2) time-resolved spectroscopic measurements revealed the photocycling rate was significantly decreased, and 3) kinetical spectroscopic measurements revealed the sensitivity of the chromophore binding Schiff base to attack by hydroxylamine was significantly increased. The QM/MM calculations show that a cavity space is present at the aromatic ring moiety in the AO3-DMP structure whereas it is absent at the corresponding ß-ionone ring moiety in the AO3-A1 structure. We discuss these alterations of the difference in interaction between the natural A1-retinal and the DMP-retinal with binding cavity residues.

Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants.

Using a quantum mechanical/molecular mechanical approach, we show the mechanisms of how the protein environment of Guillardia theta anion channelrhodopsin-1 (GtACR1) can shift the absorption wavelength. The calculated absorption wavelengths for GtACR1 mutants, M105A, C133A, and C237A are in agreement with experimentally measured wavelengths. Among 192 mutant structures investigated, mutations at Thr101, Cys133, Pro208, and Cys237 are likely to increase the absorption wavelength. In particular, T101A GtACR1 was expressed in HEK293T cells. The measured absorption wavelength is 10 nm higher than that of wild type, consistent with the calculated wavelength. (i) Removal of a polar residue from the Schiff base moiety, (ii) addition of a polar or acidic residue to the ß-ionone ring moiety, and (iii) addition of a bulky residue to increase the planarity of the ß-ionone and Schiff base moieties are the basis of increasing the absorption wavelength.

Insights into the Protein Functions and Absorption Wavelengths of Microbial Rhodopsins.

Using a quantum mechanical/molecular mechanical approach, the absorption wavelength of the retinal Schiff base was calculated based on 13 microbial rhodopsin crystal structures. The results showed that the protein electrostatic environment decreases the absorption wavelength significantly in the cation-conducting rhodopsin but only slightly in the sensory rhodopsin. Among the microbial rhodopsins with different functions, the differences in the absorption wavelengths are caused by differences in the arrangement of the charged residues at the retinal Schiff base binding moiety, namely, one or two counterions at the three common positions. Among the microbial rhodopsins with similar functions, the differences in the polar residues at the retinal Schiff base binding site are responsible for the differences in the absorption wavelengths. Counterions contribute to an absorption wavelength shift of 50-120 nm, whereas polar groups contribute to a shift of up to ∼10 nm. It seems likely that protein function is directly associated with the absorption wavelength in microbial rhodopsins.

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool.

Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2" from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.

Loss-of-function of an Arabidopsis NADPH pyrophosphohydrolase, AtNUDX19, impacts on the pyridine nucleotides status and confers photooxidative stress tolerance.

The levels and redox states of pyridine nucleotides, such as NADP(H), regulate the cellular redox homeostasis, which is crucial for photooxidative stress response in plants. However, how they are controlled is poorly understood. An Arabidopsis Nudix hydrolase, AtNUDX19, was previously identified to have NADPH hydrolytic activity in vitro, suggesting this enzyme to be a regulator of the NADPH status. We herein examined the physiological role of AtNUDX19 using its loss-of-function mutants. NADPH levels were increased in nudx19 mutants under both normal and high light conditions, while NADP+ and NAD+ levels were decreased. Despite the high redox states of NADP(H), nudx19 mutants exhibited high tolerance to moderate light- or methylviologen-induced photooxidative stresses. This tolerance might be partially attributed to the activation of either or both photosynthesis and the antioxidant system. Furthermore, a microarray analysis suggested the role of ANUDX19 in regulation of the salicylic acid (SA) response in a negative manner. Indeed, nudx19 mutants accumulated SA and showed high sensitivity to the hormone. Our findings demonstrate that ANUDX19 acts as an NADPH pyrophosphohydrolase to modulate cellular levels and redox states of pyridine nucleotides and fine-tunes photooxidative stress response through the regulation of photosynthesis, antioxidant system, and possibly hormonal signaling.

Anorectal melanoma: successful treatment by surgical excision and combination chemoimmunotherapy.

Anorectal melanoma is an extremely rare malignancy, and has a poor prognosis mainly due to delays in diagnosis and lack of effective systemic therapy. We report the case of a 63-year-old female patient with anorectal melanoma. Diagnosis was established after surgery by histology and immunohistochemistry. Surgical management consisted of abdominoperineal resection of the rectum. Postoperatively, the patient received combination therapy of dacarbazine, nimustine hydrochloride, vincristine sulfate, and interferon-beta for 3 cycles. Ten months later, a solitary brain metastatic tumor was noted in the left occipital region, which was resected surgically followed by the above combination therapy for 2 cycles. The last metastatic work-up was normal, and no evidence of recurrence was observed at 2-year follow-up. In our case, abdominoperineal resection of the rectum appears to have some effect in preventing regional and lymph node recurrence. Furthermore, our case suggests that prolongation of survival may depend on extensive block resection and combination therapy of DAV and interferon-beta.