Effect of electrostatic interaction on fibril formation of human calcitonin as studied by high resolution solid state 13C NMR

Fibrillation of a human calcitonin mutant (hCT) at the position of Asp(15) (D15N-hCT) was examined to reveal the effect of the electrostatic interaction of Asp(15) with charged side chains. The secondary structures of fibrils and soluble monomers in the site-specific (13)C-labeled D15N-hCTs were determined using (13)C cross-polarization magic angle spinning and dipolar decoupled magic angle spinning NMR approaches, sensitive to detect (13)C signals from the fibril and the soluble monomer, respectively. The local conformations and structures of D15N-hCT fibrils at pH 7.5 and 3.2 were found to be similar to each other and those of hCT at pH 3.3 and were interpreted as a mixture of antiparallel and parallel beta-sheets, whereas they were different from the hCT fibril at pH 7.5 whose structure is proposed to be antiparallel beta-sheets. Thus the negatively charged Asp(15) in the hCT molecule turned out to play an essential role in determining the structures and orientations of the hCT molecules. Fibrillation kinetics of D15N-hCT was analyzed using a two-step autocatalytic reaction mechanism. The results indicated that the replacement of Asp(15) with Asn(15) did not reduce the rate constants of the fibril formation but rather increased the rate constants at neutral pH.     

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.     

Dynamics and orientation of transmembrane peptide from bacteriorhodopsin incorporated into lipid bilayer as revealed by solid state (31)P and (13)C NMR spectroscopy.

13C and (31)P NMR spectra of a transmembrane peptide, [1-(13)C]Ala(14)-labeled A(6-34), of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine (DMPC) bilayer were recorded to clarify its dynamics and orientation in the lipid bilayer. This peptide is shown to take an alpha-helical form both in liquid crystalline and gel phases, as viewed from the conformation dependent (13)C chemical shifts. In addition, this peptide undergoes rapid rigid-body rotation about the helical axis at ambient temperature as viewed from the axially symmetric (13)C chemical shift anisotropy, whereas this symmetric anisotropy is changed to an asymmetric pattern at temperatures below 10 degrees C. We further incorporated the peptide into the spontaneously aligned DMPC bilayer to applied magnetic field, induced by dynorphin (dynorphin:DMPC =1:10), a heptadeca-opioid peptide with very high affinity to opioid receptor, in order to gain insight into its orientation in the bilayer. This magnetically aligned system turned out to be persistent even at 0 degrees C as viewed from (31)P NMR spectra of the lipid bilayer, after this peptide was incorporated into this system [A(6-34): dynorphin: DMPC = 4:10:100]. It was found from the (13)C NMR spectra of [1-(13)C]Ala(14) A(6-34) that the helical axis of A(6-34) is oriented parallel to the bilayer normal irrespective of the presence or absence of reorientation motion about the helical axis at a temperature above the lowered gel to liquid crystalline phase transition.     

NMR crystallography: the effect of deuteration on high resolution 13C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the C delta, C gamma 1, and C gamma 2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1) decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.     

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

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.     

13C and 1H solid state NMR investigation of hydration effects on gluten dynamics

The effect of hydration on the molecular dynamics of soft wheat gluten was investigated by solid state NMR. For this purpose, we recorded static and MAS ¹H spectra and SPE, CP, and other selective ¹³C spectra under MAS and dipolar decoupling conditions on samples of dry and H₂O and D₂O hydrated gluten. Measurements of carbon-proton CP times and several relaxation times (proton T₁, T_1ρ and T₂, and carbon T₁) were also performed. The combination of these techniques allowed both site-specific and domain-averaged motional information to be obtained in different characteristic frequency ranges. Domains with different structural and dynamic behaviour were identified and the changes induced by hydration on the dynamics of different domains could be monitored. The proton spin diffusion process was exploited to get information on the degree of mixing among different gluten domains. The results are consistent with the “loop and train” model proposed for hydrated gluten.     

Use of solid and gel state 13C NMR spectroscopy for differentiation between agarophytes and carrageenophytes

Both solid state (CP-MAS) and gel state (using standard solution state conditions) ¹³C NMR spectroscopy have been used to characterize a range of red algae that produce either agar or carrageenan. These techniques allow rapid determination of phycocolloid type within the algal tissue before extensive and time-consuming extractions and fractionations are carried out.The gel state technique can be used on living or dried material. Gel state spectra give high resolution and, because of the expectation that they will be correlated with the extractable phycocolloid, provide promise of a powerful technique for screening potentially useful red algae.     

NMR local range investigations in amorphous starchy substrates: II-Dynamical heterogeneity probed by (1)H/(13)C magnetization transfer and 2D WISE solid state NMR

In the preceding paper, we have investigated the structural heterogeneous character of a series of amorphous samples prepared from various starchy substrates (native potato starch, amylopectin and amylose) following different techniques of preparation (casting, freeze drying and solvent exchange). Spectral decompositions of the C1 resonances of the (13)C CP-MAS (Cross Polarization and Magic Angle Spinning) spectra under (1)H decoupling have shown the existence of five main types of alpha(1-4) linkages. In this part, 2D solid state NMR WISE experiments and the (13)C/(1)H magnetization transfer in CP as a local probe for both structures and dynamics were used. The (13)C CP magnetization curves versus contact time of each C1 component in each recorded spectrum were fitted with an analytic function taking into account two (1)H reservoirs. Interpretation of the characteristic times derived from fitting yields some improvements on the knowledge of the heterogeneity of the samples and on the water molecules distribution.     

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.     

Partial site-specific assignment of a uniformly (13)C, (15)N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz (15)N-(13)CO-(13)Calpha and (15)N-(13)Calpha-(13)CX correlation spectra, using uniformly (13)C, (15)N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic (15)N-(13)Calpha-(13)Cbeta side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively (13)C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Calpha and Cbeta resonances indicated the presence of typical alpha-helical secondary structure, consistent with previous studies.     

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-¹³C,¹⁵N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical ¹³C peak line widths are 0.5 ppm, resulting in C-C and C-CO regions that exhibit many resolved peaks. Two-dimensional carbon–carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 mol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.     

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.     

Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I.C.P. & Sprott, G.D. (1983) J. Bacteriol. 156, 316-326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.     

Crystal structure and solid state 13C NMR analysis of nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-gluco- and D-galactopyranosides

The X-ray diffraction analysis of o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside (1), m-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, p-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside was performed. It was found that except in the case of 1, all other crystals have one molecule in the independent part of the crystal unit cell. The results support the opinion that the nitro group does not conjugate effectively with the phenyl ring. In the 13C CP MAS spectrum of 1 the signals are split, confirming the presence of two independent molecules. Similarly, the 13C CP MAS NMR spectrum of p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside indicated the presence of two non-equivalent molecules in the crystal unit. One of these molecules has more conformational freedom enabling rotation of the phenyl ring.     

Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose. The fractional enrichment pattern of teicoplanin produced in the medium containing [1-(13)C]glucose was obtained from a one-dimensional (13)C spectrum. The enrichment pattern showed characteristic peaks indicating that amino acids 3 and 7 are derived from acetate, whereas amino acids 1, 2, 4, 5, and 6 are derived from tyrosine. Multiplet structures in heteronuclear single quantum coherence spectra of teicoplanin produced in the medium containing [U-(13)C]glucose showed characteristic coupling patterns supporting these results. Fractional enrichment patterns and multiplet structures of the three sugars in teicoplanin showed that about 50% of the sugars have the same labeling pattern as the glucose substrate whereas the rest have a labeling pattern showing that they are reassembled, probably from precursors in the primary metabolism.     

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully¹³ C/¹⁵N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue¹³ C-¹⁵N correlation experiments with¹³ C-¹³C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using ¹⁵N instead of¹³ C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the ¹⁵N and ¹³C resonances in human ubiquitin.     

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.