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.     

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.     

Secondary structure and backbone dynamics of Escherichia coli diacylglycerol kinase,as revealed by site-directed solid-state 13C NMR

To gain insight into secondary structure and backbone dynamics, we have recorded ¹³C NMR spectra of [3-¹³C]Ala-, [1-¹³C]Val-labeled Escherichia coli diacylglycerol kinase (DGK), using cross-polarization-magic angle spinning (CP-MAS) and single-pulse excitation with dipolar decoupled-magic angle spinning (DD-MAS) methods. DGK was either solubilized in n-decyl-β-maltoside (DM) micelle or integrated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Surprisingly, the ¹³C NMR spectra were broadened to yield rather featureless peaks at physiological temperatures, both in DM solution or lipid bilayers at liquid crystalline phase, due to interference of motional frequencies of DGK with frequencies of magic angle spinning (MAS) or proton decoupling (10⁴ or 10⁵ Hz, respectively). In gel phase lipids, however, up to six distinct ¹³C NMR peaks were well-resolved due to lowered fluctuation frequencies (<10⁵ Hz) for the transmembrane region, the amphipathic α-helices and loops. While DGK can be tightly packed in gel phase lipids, DGK is less tightly packed at physiological temperatures, where it becomes more mobile. The fact that the enzymatic activity is low under conditions where motion is restricted and high when conformational fluctuations can occur suggests that acquisition of low frequency backbone motions, on the microsecond to millisecond time scale, may facilitate the efficient enzymatic activity of DGK.     

Dynamic pictures of membrane proteins in two-dimensional crystal, lipid bilayer and detergent as revealed by site-directed solid-state 13C NMR

We have compared site-directed 13C solid-state NMR spectra of [3-13C]Ala- and/or [1-13C]Val-labeled membrane proteins, including bacteriorhodopsin (bR), pharaonis phoborhodopin (ppR), its cognate transducer (pHtrII) and Escherichia coli diacylglycerol kinase (DGK), in two-dimensional (2D) crystal, lipid bilayers, and detergent. Restricted fluctuation motions of these membrane proteins due to oligomerization of bR by specific protein-protein interactions in the 2D crystalline lattice or protein complex between ppR and pHtrII provide the most favorable environment to yield well-resolved, fully visible 13C NMR signals for [3-13C]Ala-labeled proteins. In contrast, several signals from such membrane proteins were broadened or lost owing to interference of inherent fluctuation frequencies (10(4)-10(5)Hz) with frequency of either proton decoupling or magic angle spinning, if their 13C NMR spectra were recorded as a monomer in lipid bilayers at ambient temperature. The presence of such protein dynamics is essential for the respective proteins to achieve their own biological functions. Finally, spectral broadening found for bR and DGK in detergents were discussed.     

A (13)C NMR study on [3-(13)C]-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

We have recorded (13)C NMR spectra of selectively [3-(13)C]Ala-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled synthetic transmembrane peptides of bacteriorhodopsin (bR) and enzymatically cleaved C-2 fragment in the solid and dimyristoylphosphatidylcholine bilayer. It turned out that these transmembrane peptides either in hexafluoroisopropanol or cast from it take an ordinary alpha-helix (alpha(I)-helix) irrespective of their amino acid sequences with reference to the conformation-dependent (13)C chemical shifts of (Ala)(n) taking the alpha-helix form. These transmembrane peptides are not always static in the lipid bilayer as in the solid state but undergo rigid-body motions with various frequencies as estimated from suppressed peaks either by fast isotropic or large-amplitude motions (>10(8) Hz) or intermediate frequencies (10(5) or 10(3) Hz). Further, (13)C chemical shifts of the [3-(13)C]Ala-labeled peptides in the bilayer were displaced downfield by 0.3-1.1 ppm depending upon amino acid sequence with respect to those in the solid state, which were explained in terms of local conformational fluctuation (10(2) Hz) deviated from the torsion angles (alpha(II)-helix) from those of standard alpha-helix, under anisotropic environment in lipid bilayer, in addition to the above-mentioned rigid-body motions. The carbonyl (13)C peaks, on the other hand, are not sensitively displaced by such local anisotropic fluctuations, because they are more sensitive to the manner of hydrogen-bond interactions. The amino acid sequences of these peptides inserted within the bilayer were not always the same as those of intact bR, causing disposition of the transmembrane alpha-helical segment from that of intact bR. Finally, we confirmed that the (13)C NMR peak positions of the random coil form are located at the boundary between the alpha-helix and a turned structure in loop regions.     

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR

13C NMR spectra of [3-(13)C]Ala- and [1-(13)C]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 alphaII-helices. Surprisingly, the 13C NMR spectra of [3-(13)C]Ala-D85N turned out to be very similar to those of [3-(13)C]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 alphaII-helices of the M-like state are suppressed already by fluctuation motions in the order of 10(4)-10(5) Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic alpha-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 alpha-helices followed by Pro residues in [1-(13)C]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-(13)C]Val and [3-(13)C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Glutamic acid residues of bacteriorhodopsin at the extracellular surface as determinants for conformation and dynamics as revealed by site-directed solid-state 13C NMR

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal alpha-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane alpha-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(-4)-10(-5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-A depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 micro M Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane.     

Conformation and dynamics of the [3-(13)C]Ala, [1-(13)C]Val-labeled truncated pharaonis transducer, pHtrII(1-159), as revealed by site-directed (13)C solid-state NMR: changes due to association with phoborhodopsin (sensory rhodopsin II)

We have recorded (13)C NMR spectra of the [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis transducer pHtrII(1-159) in the presence and absence of phoborhodopsin (ppR or sensory rhodopsin II) in egg phosphatidylcholine or dimyristoylphosphatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR. Two kinds of (13)C NMR signals of [3-(13)C]Ala-pHtrII complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR. One of these resonances was at the peak position of the low-field alpha-helical peaks (alpha(II)-helix) and is identified with cytoplasmic alpha-helices protruding from the bilayers; the other was the high-field alpha-helical peak (alpha(I)-helix) and is identified with the transmembrane alpha-helices. The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR. This is caused by an increased fluctuation frequency of the cytoplasmic alpha-helix from 10(5) Hz in the uncomplexed states to >10(6) Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (10(5) Hz), as viewed from the (13)C NMR spectra of [3-(13)C]Ala-labeled pHtrII. Consistent with this view, the (13)C DD-MAS NMR signals of the cytoplasmic alpha-helices of the complexed [3-(13)C]Ala-pHtrII in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0 degrees C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the (13)C CP-MAS spectra of [1-(13)C]Val-labeled complexed pHtrII showed that the (13)C NMR signals of the transmembrane alpha-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane alpha-helices from 10(3) Hz of the uncomplexed states to 10(4) Hz of the complexed states. These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR-pHtrII complex is not aggregated. We show that (13)C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state 13C NMR studies on [3-13C]Ala- and [1-13C]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 alpha-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 10(4)-10(5) 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 degrees 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.     

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.     

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

Regio-selective detection of dynamic structure of transmembrane alpha-helices as revealed from (13)C NMR spectra of [3-13C]Ala-labeled bacteriorhodopsin in the presence of Mn2+ ion.

13C Nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane alpha-helices by using NMR relaxation reagent, Mn(2+) ion. As a result of selective suppression of (13)C NMR signals from the surfaces in the presence of Mn(2+) ions, several (13)C NMR signals of Ala residues in the transmembrane alpha-helices were identified on the basis of site-directed mutagenesis without overlaps from (13)C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between (13)C nucleus in bR and Mn(2+) ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn(2+) ion was estimated as 8.7 A to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C(beta) (13)C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of (13)C NMR spectra of bR in the presence of Mn(2+) ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane alpha-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane alpha-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp 85 and deprotonation of Schiff base as studied by 13C NMR

According to previous X-ray diffraction studies, the D85N mutant of bacteriorhodopsin (bR) with unprotonated Schiff base assumes a protein conformation similar to that in the M photointermediate. We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N and D85N/D96N mutants at ambient temperature to examine how conformation and dynamics of the protein backbone are altered when the Schiff base is protonated (at pH 7) and unprotonated (at pH 10). Most notably, we found that the peak intensities of three to four [3-(13)C]Ala-labeled residues from the transmembrane alpha-helices, including Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D85N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-)(5) or 10(-)(4) s, which interferes with proton decoupling frequency or frequency of magic angle spinning, respectively, essential for an attempted peak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of transmembrane helices upon deprotonation of Schiff base and the formation of the M-like state in the absence of illumination. The spectra detected more rapid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-)(4) to 10(-)(5) s. Conformational changes in the transmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-(13)C-labeled Val 49 (helix B), 69 (B-C loop), and [3-(13)C]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side substantially modulated the conformation and dynamics of the extracellular region through long-distance interaction.     

13C-NMR Study of propionate metabolism by sludges from bioreactors treating sulfate and sulfide rich wastewater

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution¹³ C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented.¹³ C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-¹³C]propionate into [2-¹³C]propionate and the formation of acetate equally labeled in the C₁ and C₂ position. In the absence of sulfate, [3-¹³C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-¹³C]propionate partly into the higher fatty acid 2-methyl[2,3-¹³C]butyrate during the simultaneous degradation of [2,3-¹³C]propionate and butyrate. [4,5-¹³C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.     

Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR

By applying [1-¹³C]- and [2-¹³C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 Å to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region β1-β2 but also for β-strands β1, β2, β3, and β5 as well as for the loop connecting α1 and β3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.