Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.     

Association of pharaonis phoborhodopsin with its cognate transducer decreases the photo-dependent reactivity by water-soluble reagents of azide and hydroxylamine

pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.     

Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.     

Time-resolved WAXS reveals accelerated conformational changes in iodoretinal-substituted proteorhodopsin

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.     

C nuclear magnetic resonance observations on the interaction between p-amidinophenylpyruvic acid and trypsin

The formation of an enzyme-inhibitor adduct between bovine trypsin and [2-¹³C]p-amidinophenylpyruvic acid has been investigated by ¹³C NMR spectroscopy. The observation of a resonance at 100.8 ppm demonstrates that the hemiketal formed between the hydroxyl of serine-195 and the 2-¹³C carbon of p-amidinophenylpyruvic acid is sp³ hybridized with no significant deviation from tetrahedral geometry. It is shown that stabilization of the hemiketal oxyanion if it occurs is less effective than in chloromethylketone inhibitor complexes. The tetrahedral adduct is stable from pH 3 to 8. The mechanisms of breakdown of the tetrahedral adduct at pH extremes are discussed.     

Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by C NMR

We have recorded ¹³C NMR spectra of [3-¹³C]-, [1-¹³C]Ala-, and [1-¹³C]Val-labeled bacteriorhodopsin (bR), W80L and W12L mutants and bacterio-opsin (bO) from retinal-deficient E1001 strain, in order to examine the possibility of their millisecond to microsecond local fluctuations with correlation time in the order of 10⁻⁴ to 10⁻⁵ s, induced or prevented by disruption or assembly of two-dimensional (2D) crystalline lattice, respectively, at ambient temperature. The presence of disrupted or disorganized 2D lattice for W12L, W80L and bO from E1001 strain was readily visualized by increased relative proportions of surrounding lipids per protein, together with their broadened ¹³C NMR signals of transmembrane α-helices and loops in [3-¹³C]Ala-labeled proteins, with reference to those of wild-type. In contrast, ¹³C CP-MAS NMR spectra of [1-¹³C]Ala- and Val-labeled these mutants were almost completely suppressed, owing to the presence of fluctuations with time scale of 10⁻⁴ s interfered with magic angle spinning. In particular, ¹³C NMR signals of [1-¹³C]Ala-labeled transmembrane α-helices of wild-type were almost completely suppressed at the interface between the surface and inner part (up to 8.7 Å deep from the surface) with reference to those of the similarly suppressed peaks by Mn²⁺-induced accelerated spin-spin relaxation rate. Such fluctuation-induced suppression of ¹³C NMR peaks from the interfacial regions, however, was less significant for [1-¹³C]Val-labeled proteins, because fluctuation motions in Val residues with bulky side-chains at the C_α moiety were modified to those of longer correlation time (>10⁻⁴ s), if any, by residue-specific manner. To support this view, we found that such suppressed ¹³C NMR signals of [1-¹³C]Ala-labeled peaks in the wild-type were recovered for D85N and bO in which correlation times of fluctuations were shifted to the order of 10⁻⁵ s due to modified helix-helix interactions as previously pointed out [Biochemistry, 39 (2000) 14472; J. Biochem. (Tokyo) 127 (2000) 861].     

Solid-state structure of N-o-, N-m-, and N-p-nitrophenyl-2,3,4-tri-O-acetyl-β-d-xylopyranosylamines

Comprehensive structural analyses were performed for N-o-, N-m-, and N-p-nitrophenyl-2,3,4-tri-O-acetyl-β-d-xylopyranosylamines. Single-crystal X-ray diffraction data were collected and revealed that one compound under investigation undergoes temperature-dependent polymorph transitions (crystal structures of three polymorphs were obtained). The number of molecules in the independent part of the crystal unit cells was in agreement with the number of resonances in solid-state ¹³C NMR spectra. Therefore, the compounds exist as single polymorphs at room temperature, as confirmed by powder X-ray diffraction measurements. Significant differences in ¹³C chemical shifts between solution and solid-state NMR for selected carbon atoms confirmed the existence of intra- and/or intermolecular interactions.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O177

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the ¹H and ¹³C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.     

Spectral editing of alanine,serine, and threonine in uniformly labeled proteins based on frequency-selective homonuclear recoupling in solid-state NMR

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit ¹³C-¹³C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077–8083]. The signals of alanine, serine, or threonine residues are selected out by selective ¹³Cα-¹³Cβ double-quantum filtering (DQF). The ¹³Cα-¹³Cβ correlations of alanine residues are selectively established with efficiency up to?~?1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly ¹³C, ¹⁵N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using ¹H, ¹³C, and ³¹P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→     

C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevulinic acid destined for chlorophyll formation in dark-grown Scenedesmus obliquus

The ¹³C-nuclear magnetic resonance (NMR) spectra of chlorophyll a formed in dark-grown Scenedesmus obliquus (Turp.) Kützing in the presence of [1-¹³C]glutamate, [2-¹³C]- and [1-¹³C]glycineshowed that the ¹³C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of chlorophyll a. No specific enrichment of these nine carbon atoms was observed in the spectrum of chlorophyll a formed in the presence of [1-¹³C]-glycine. These labeling patterns provide evidence for the operation of the C₅-pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Structures of chlorosomes and aggregated BChlc inChlorobium tepidum from solid state high resolution CP/MAS13C NMR

Cross polarization/magic angle spinning (CP/MAS)¹³C (solid state high resolution) NMR spectra were observed for chlorosomes and BChlc aggregates. Similarity of both kinds of spectra (except for some signals assignable to proteins and lipids in chlorosomes) indicates that BChlc's in chlorosomes are present just as in synthetic BChlc aggregates. Chemical shifts for C13¹ carbonyl and C3¹ hydroxylethyl carbons indicate hydrogen bonding between them. Comparison of solution and solid state¹³C NMR chemical shifts shows the five coordinated nature of BChlc aggregates. Some chemical shift differences were attributable to ring currents shifts. Their comparisons with calculated ring current shift values predicted structures for the aggregates. Cross polarization dynamics of the CP/MAS¹³C NMR signals explored dynamic and structural nature of the BChlc aggregates.     

Significance of low-frequency local fluctuation motions in the transmembrane B and C α-helices of bacteriorhodopsin,to facilitate efficient proton uptake from the cytoplasmic surface,as revealed by site-directed solid-state <Superscript>13</Superscript>C NMR

¹³C NMR spectra of [1-¹³C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane -helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10⁴ Hz in the transmembrane B -helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed ¹³C NMR signal of the [1-¹³C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the ¹³C NMR of [1-¹³C]-labeled Pro50. This means that the transmembrane B -helix is able to acquire the fluctuation motion with a frequency of 10⁴ Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10⁴ Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the ¹³C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced pK_a value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C -helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the ¹³C NMR data of the D85N/V49A mutant.     

The 13C NMR spectra of some rosane diterpenoids

The ¹³C NMR signals of rosenonolactone have been assigned utilizing the ¹³C-¹³C couplings in material obtained biosynthetically from sodium [1,2-¹³C₂] acetate.     

Structure of the O-Specific polysaccharide from Shewanella japonica KMM 3601 containing 5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid

Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with ¹H and ¹³C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: →4)-α-4eLegp5Ac7Ac-(2→4)-β-d-GlcpA3Ac-(1→3)-β-d-GalpNAc-(1→.     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori

Summary. It was observed by solution-state ¹³C NMR spectroscopy that a great portion of the ¹³C of [1-¹³C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-¹³C]Glycine was detected along with [1-¹³C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-¹³C]serine. This formation of [1-¹³C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state ¹³C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [¹³C]formate revealed that serine C3 was labelled specifically with ¹³C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine. Received May 4, 1999 Accepted December 10, 1999