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.

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.

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.

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.

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.

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.

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.

Probing the Cl--pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle.

Halorhodopsin (HR) functions as a light-driven inward Cl- pump. The Cl- transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl- was added to unphotolyzed Cl--free NpHR-Brub complex, Brub caused the absorption spectral change in response to the Cl- binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl--puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR'. These results indicate that Cl- is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR' still maintains an altered protein conformation while another Cl- already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N-O quasi-equilibrium toward N, supporting Cl- release during the N to O transition. The membrane potential had a much larger effect on the Cl- transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl- transfers during N to O and O to NpHR' transitions.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

Role of Thr218 in the light-driven anion pump halorhodopsin from Natronomonas pharaonis.

Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (P(i), i = 1-4) were adequate to describe the photocycle. The third component, P3, was a quasi-equilibrium complex between the N and O intermediates, where a N ↔ O + Cl⁻ equilibrium was attained. The K(d,N↔O) values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, ΔV(N→O), were estimated from the pressure dependence of K(d,N↔O). A comparison between K(d,N↔O) and ΔV(N→O) led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K(d,N↔O). For some mutants, however, large ΔV(N→O) values were found, whereas the K(d,N↔O) values were small. This suggests that the special coordination of a water molecule with the OH group of Thr is necessary for the increase in K(d,N↔O). Mutants with a small K(d,N↔O) showed low pumping activities in the presence of inside negative membrane potential, while the mutant activities were not different in the absence of membrane potential. The effect of the mutation on the pumping activities is discussed.

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.

Homotrimer formation and dissociation of pharaonis halorhodopsin in detergent system.

Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.

Photoinduced proton release in proteorhodopsin at low pH: the possibility of a decrease in the pK(a) of Asp227.

Proteorhodopsin (PR) is one of the microbial rhodopsins that are found in marine eubacteria and likely functions as an outward light-driven proton pump. Previously, we [Tamogami, J., et al. (2009) Photochem. Photobiol.85, 578-589] reported the occurrence of a photoinduced proton transfer in PR between pH 5 and 10 using a transparent ITO (indium-tin oxide) or SnO(2) electrode that works as a time-resolving pH electrode. In the study presented here, the proton transfer at low pH (<4) was investigated. Under these conditions, Asp97, the primary counterion to the protonated Schiff base, is protonated. We observed a first proton release that was followed by an uptake; during this process, however, the M intermediate did not form. Through the use of experiments with several PR mutants, we found that Asp227 played an essential role in proton release. This residue corresponds to the Asp212 residue of bacteriorhodopsin, the so-called secondary Schiff base counterion. We estimated the pK(a) of this residue in both the dark and the proton-releasing photoproduct to be ~3.0 and ~2.3, respectively. The pK(a) value of Asp227 in the dark was also estimated spectroscopically and was approximately equal to that determined with the ITO experiments, which may imply the possibility of the release of a proton from Asp227. In the absence of Cl(-), we observed the proton release in D227N and found that Asp97, the primary counterion, played a key role. It is inferred that the negative charge is required to stabilize the photoproducts through the deprotonation of Asp227 (first choice), the binding of Cl(-) (second choice), or the deprotonation of Asp97. The photoinduced proton release (possibly by the decrease in the pK(a) of the secondary counterion) in acidic media was also observed in other microbial rhodopsins with the exception of the Anabaena sensory rhodopsin, which lacks the dissociable residue at the position of Asp212 of BR or Asp227 of PR and halorhodopsin. The implication of this pK(a) decrease is discussed.

Influence of halide binding on the hydrogen bonding network in the active site of Salinibacter sensory rhodopsin I.

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.

Ligand specificity determined by differentially arranged common ligand-binding residues in bacterial amino acid chemoreceptors Tsr and Tar.

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.

Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state.

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.

An active photoreceptor intermediate revealed by in situ photoirradiated solid-state NMR spectroscopy.

A novel, to our knowledge, in situ photoirradiation system for solid-state NMR measurements is improved and demonstrated to successfully identify the M-photointermediate of pharaonis phoborhodopsin (ppR or sensory rhodopsin II), that of the complex with transducer (ppR/pHtrII), and T204A mutant embedded in a model membrane. The (13)C NMR signals from [20-(13)C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates with 13-cis, 15-anti retinal configuration coexisted under the continuously photoirradiated condition. NMR signals observed from the photoactivated retinal provide insights into the process of photocycle in the ppR/pHtrII complex.

Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis.

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ∼500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.