Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization.

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

Organoruthenium-catalyzed chemical protein synthesis to elucidate the functions of epigenetic modifications on heterochromatin factors.

The application of organometallic compounds for protein science has received attention. Recently, total chemical protein synthesis using transition metal complexes has been developed to produce various proteins bearing site-specific posttranslational modifications (PTMs). However, in general, significant amounts of metal complexes were required to achieve chemical reactions of proteins bearing a large number of nucleophilic functional groups. Moreover, syntheses of medium-size proteins (>20 kDa) were plagued by time-consuming procedures due to cumbersome purification and isolation steps, which prevented access to variously decorated proteins. Here, we report a one-pot multiple peptide ligation strategy assisted by an air-tolerant organoruthenium catalyst that showed more than 50-fold activity over previous palladium complexes, leading to rapid and quantitative deprotection on a protein with a catalytic amount (20 mol%) of the metal complex even in the presence of excess thiol moieties. Utilizing the organoruthenium catalyst, heterochromatin factors above 20 kDa, such as linker histone H1.2 and heterochromatin protein 1α (HP1α), bearing site-specific PTMs including phosphorylation, ubiquitination, citrullination, and acetylation have been synthesized. The biochemical assays using synthetic proteins revealed that the citrullination at R53 in H1.2 resulted in the reduced electrostatic interaction with DNA and the reduced binding affinity to nucleosomes. Furthermore, we identified a key phosphorylation region in HP1α to control its DNA-binding ability. The ruthenium chemistry developed here will facilitate the preparation of a variety of biologically and medically significant proteins containing PTMs and non-natural amino acids.

Effects of Glutamate Arginylation on α-Synuclein: Studying an Unusual Post-Translational Modification through Semisynthesis.

A variety of post-translational modifications (PTMs) are believed to regulate the behavior and function of α-synuclein (αS), an intrinsically disordered protein that mediates synaptic vesicle trafficking. Fibrils of αS are implicated in neurodegenerative disorders such as Parkinson's disease. In this study, we used chemical synthesis and biophysical techniques to characterize the neuroprotective effects of glutamate arginylation, a hitherto little characterized PTM in αS. We developed semisynthetic routes combining peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation (NCL) to site-specifically introduce the PTM of interest along with fluorescent probes into αS. We synthesized the arginylated glutamate as a protected amino acid, as well as a novel ligation handle for NCL, in order to generate full-length αS modified at various individual sites or a combination of sites. We assayed the lipid-vesicle binding affinities of arginylated αS using fluorescence correlation spectroscopy (FCS) and found that arginylated αS has the same vesicle affinity compared to control protein, suggesting that this PTM does not alter the native function of αS. On the other hand, we studied the aggregation kinetics of modified αS and found that arginylation at E83, but not E46, slows aggregation and decreases the percentage incorporation of monomer into fibrils in a dose-dependent manner. Arginylation at both sites also resulted in deceleration of fibril formation. Our study represents the first synthetic strategy for incorporating glutamate arginylation into proteins and provides insight into the neuroprotective effect of this unusual PTM.

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

Chemical Synthesis of Cys-Containing Protein via Chemoselective Deprotection with Different Palladium Complexes.

We report selective removals of N-terminal and internal Cys protecting groups using different palladium complexes to facilitate the efficient chemical protein synthesis. Utilizing the orthogonal deprotection pairs, we accomplished chemical synthesis of histone H3 containing trimethylated Lys through the combination of Pd(0)-mediated Alloc deprotection for one-pot multiple peptide ligation and Pd(II)Cl2-mediated Acm deprotection to recover native Cys residues after desulfurization.

Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin.

Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the "monomerization effects": Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.

Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.

Mastigocladopsis repens halorhodopsin (MrHR) is a Cl--pumping rhodopsin that belongs to a distinct cluster far from other Cl- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I- similar to Cl- but cannot transport it. I--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl--pumping photocycle, Cl- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl--binding affinity nor the absorption spectrum, but completely eliminated the Cl--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H+ release was detected for photolyzed MrHR by using an indium­tin oxide electrode, which acts as a good time-resolved pH sensor. This H+ release disappeared in the I--bound form of the wild-type and Cl--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl- movement to the CP channel.

Triple Function of 4-Mercaptophenylacetic Acid Promotes One-Pot Multiple Peptide Ligation.

One-pot multiple peptide ligation is a key technology to improve the efficiency of chemical protein synthesis. One-pot repetitive peptide ligation requires a cycle of three steps: peptide ligation, removal of a protecting group, and inactivation of the deprotection reagent. However, previous strategies are not sufficient because of harsh deprotection conditions, slow deprotection rates, and difficulty in quenching the deprotection reagent. To address these issues, we developed a rapid, efficient deprotection and subsequent quenching strategy using an allyloxycarbonyl group to protect the N-terminal cysteine residue. 4-Mercaptophenylacetic acid (MPAA), a thiol additive for native chemical ligation, functioned not only as a scavenger for π-allyl palladium complexes, but also as a quencher of palladium(0) complexes. By utilizing the multifunctionality of MPAA, we carried out a one-pot five-segment ligation to afford histone H2AX (142 amino acids), which was isolated in 59 % yield.

Low-temperature Raman spectroscopy reveals small chromophore distortion in primary photointermediate of proteorhodopsin.

Proteorhodopsin (PR) is a microbial rhodopsin functioning as a light-driven proton pump in aquatic bacteria. We performed low-temperature Raman measurements of PR to obtain the structure of the primary photoproduct, the K intermediate (PRK ). PRK showed the hydrogen-out-of-plane modes that are much less intense than those of bacteriorhodopsin as the prototypical light-driven proton pump from haloarchaea. The present results reveal the significantly relaxed chromophore structure in PRK , which can be coupled to the slow kinetics of the K intermediate. This structure suggests that PR transports protons using the small energy storage within the chromophore at the start of its photocycle.

Retinal Configuration of ppR Intermediates Revealed by Photoirradiation Solid-State NMR and DFT.

Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N'-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N'-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at -60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N'- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N'-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N'-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N'-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.

Efficient peptide ligation between allyl-protected Asp and Cys followed by palladium-mediated deprotection.

An efficient method for peptide ligation between C-terminal Asp(OAllyl) and N-terminal Cys has been developed. Peptide ligation and removal of the allyl group at the Asp carboxylate side chain proceeded in one pot by adding a small amount of Pd/TPPTS complex. Based on this efficient synthetic method, PEP-19 (61 amino acids), which is highly expressed in Purkinje cells, was synthesized.

Interhelical interactions between D92 and C218 in the cytoplasmic domain regulate proton uptake upon N-decay in the proton transport of Acetabularia rhodopsin II.

Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.

Photocycle of Sensory Rhodopsin II from Halobacterium salinarum (HsSRII): Mutation of D103 Accelerates M Decay and Changes the Decay Pathway of a 13-cis O-like Species.

Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild-type > D103N > D103E; (2) altered decay pathway of a 13-cis O-like species. The 13-cis O-like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild-type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII.

Existence of two O-like intermediates in the photocycle of Acetabularia rhodopsin II, a light-driven proton pump from a marine alga.

A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M⇄N⇄O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.

Chemical synthesis of dual labeled proteins via differently protected alkynes enables intramolecular FRET analysis.

We report a novel method for multisite protein conjugation by setting differently silyl-protected alkynes as conjugation handles, which can remain intact through the whole synthetic procedure and provide sequential and orthogonal conjugation. This strategy enables efficient preparation of a dual dye-labeled protein and structural analysis via an intramolecular FRET mechanism.

Photochemical characterization of actinorhodopsin and its functional existence in the natural host.

Actinorhodopsin (ActR) is a light-driven outward H+ pump. Although the genes of ActRs are widely spread among freshwater bacterioplankton, there are no prior data on their functional expression in native cell membranes. Here, we demonstrate ActR phototrophy in the native actinobacterium. Genome analysis showed that Candidatus Rhodoluna planktonica, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells. The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia coli membranes. The purified RpActR showed λmax at 534nm and underwent a photocycle characterized by the very fast formation of M intermediate. The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+ pumps. In contrast to conventional O, the accumulation of P620 remains prominent, even at high pH. Flash-induced absorbance changes suggested that there exists only one kind of photocycle at any pH. However, above pH7, RpActR shows heterogeneity in the H+ transfer sequences: one first captures H+ and then releases it during the formation and decay of P620, while the other first releases H+ prior to H+ uptake during P620 formation.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump.

Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H(+) and Na(+) pumps and an inward Cl(-) pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H(+) pump bacteriorhodopsin, which was converted to a Cl(-) pump in 1995 by a single amino acid replacement. In this study we report the first success of the reverse conversion from a Cl(-) pump to a H(+) pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl(-) pump and belongs to a cluster that is far distant from the known Cl(-) pumps. With a single amino acid replacement, MrHR is converted to a H(+) pump in which dissociable residues function almost completely in the H(+) relay reactions. MrHR most likely evolved from a H(+) pump, but it has not yet been highly optimized into a mature Cl(-) pump.