Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Inter‐relationships between the heterotrimeric Gβ subunit AGB1, the receptor‐like kinase FERONIA,and RALF1 in salinity response

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor‐like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G‐protein‐coupled‐receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1‐like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein β subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt‐induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na⁺ accumulation and decreasing K⁺ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1‐independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.     

RALF22 promotes plant immunity and amplifies the Pep3 immune signal

Rapid alkalinization factors(RALFs) in plants have been reported to dampen pathogenassociated molecular pattern(PAMP)-triggered immunity via suppressing PAMP-induced complex formation between the pattern recognition receptor(PRR) and its co-receptor BAK1. However, the direct and positive role of RALFs in plant immunity remains largely unknown. Herein, we report the direct and positive roles of a typical RALF, RALF22, in plant immunity. RALF22alone directly elicited a variety of typical immune re...     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Solution structure of the cysteine‐rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF‐like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor‐ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand‐binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine‐rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28‐Ala70) by heteronuclear NMR, with a ¹³C‐/¹⁵N‐labeled sample. The tertiary structure of the CRD comprises a β‐sheet with two strands, followed by a 3₁₀ helix and a C‐terminal α‐helix, and is stabilized by three disulfide bonds connecting Cys36‐Cys49, Cys52‐Cys67, and Cys55‐Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1‐C2 module type), but not to the CRD of B‐cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1‐D2 module type). This is the first structural report about the A1‐C2 type CRD that could bind to the known target.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

Solution structure for an Encephalitozoon cuniculi adrenodoxin‐like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria‐like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome‐like organelles in E. cuniculi, the 128‐residue, adrenodoxin‐like protein Ec‐Adx (UniProt ID Q8SV19) in the [2Fe‐2S] ferredoxin superfamily. Oxidized Ec‐Adx contains a mixed four‐strand β‐sheet, β2‐β1‐β4‐β3 (↓↑↑↓), loosely encircled by three α‐helices and two 3₁₀‐helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin‐like proteins except for the absence of a fifth anti‐parallel β‐strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the ¹H‐¹⁵N HSQC spectrum of Ec‐Adx. These missing residues are clustered primarily in two regions, G48‐V61 and L94‐L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe‐2S] cluster. Missing amide resonances in ¹H‐¹⁵N HSQC spectra are detrimental to NMR‐based solution structure calculations because ¹H‐¹H NOE restraints are absent (glass half‐empty) and this may account for the absent β‐strand (β5) and the position of α3 in oxidized Ec‐Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe‐2S] cluster in oxidized Ec‐Adx (glass half‐full).     

Distinct solid and solution state self‐assembly pathways of RADA16‐I designer peptide

Solid state NMR measurements on selectively ¹³C‐labeled RADA16‐I peptide (COCH₃–RADARADARADARADA–NH₂) were used to obtain new molecular level information on the conversion of α‐helices to β‐sheets through self‐assembly in the solid state with increasing temperature. Isotopic labeling at the A4 C_β site enabled rapid detection of ¹³C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α‐helix and β‐strand content. These samples were then probed at room temperature for intermolecular ¹³C–¹³C nuclear dipolar couplings with the PITHIRDS‐CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X‐ray diffraction. Independence of PITHIRDS‐CT decay shapes on overall α‐helical and β‐strand content infers that β‐strands are not observed without association with β‐sheets, indicating that β‐sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS‐CT NMR data were compared with results of similar measurements on RADA16‐I nanofibers produced by self‐assembly in aqueous salt solution. We report that β‐sheets formed through self‐assembly in the solid state have a structure that differs from those formed through self‐assembly in the solution state. Specifically, solid state RADA16‐I self‐assembly produces in‐register parallel β‐sheets, whereas nanofibers are composed of stacked parallel β‐sheets with registry shifts between adjacent β‐strands in each β‐sheet. These results provide evidence for environment‐dependent self‐assembly mechanisms for RADA16‐I β‐sheets as well as new constraints on solid state self‐assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of five RALF-like genes from<Emphasis Type="Italic"> Solanum chacoense</Emphasis> provides support for a developmental role in plants

Five RALF (rapid alkalinization factor)-like genes, named ScRALF1 to 5, were isolated from fertilized ovule and ovary cDNA libraries of Solanum chacoense. They showed high sequence similarities with the RALF protein sequence from Nicotiana tabacum, and exhibited the characteristic architecture of RALF polypeptides. All ScRALFs were moderately to highly expressed at some stage of fruit maturation. ScRALF1 and ScRALF3 were predominantly expressed in ovaries and larger fruits, while ScRALF2, ScRALF4, and ScRALF5 were also expressed in other tissues, indicating that while some RALFs may be involved in fruit maturation, others could be involved in other developmental processes. Wounding or treatment of plants with growth regulators involved in plant defense responses had no significant impact on the mRNA level of any of these genes. These results suggest and support previous data showing that RALF peptides are more likely to act as a small peptide involved in plant development than in defense responses.GenBank accession numbers: ScRALF1, AY422824; ScRALF2, AY422825; ScRALF3, AY422826; ScRALF4, AY422827; ScRALF5, AY655141     

Amino acid transporter mutants of Arabidopsis provides evidence that a non‐mycorrhizal plant acquires organic nitrogen from agricultural soil

Although organic nitrogen (N) compounds are ubiquitous in soil solutions, their potential role in plant N nutrition has been questioned. We performed a range of experiments on Arabidopsis thaliana genetically modified to enhance or reduce root uptake of amino acids. Plants lacking expression of the Lysine Histidine Transporter 1 (LHT1) displayed significantly lower contents of ¹³C and ¹⁵N label and of U‐¹³C₅,¹⁵N₂ L‐glutamine, as determined by liquid chromatography–mass spectrometry when growing in pots and supplied with dually labelled L‐glutamine compared to wild type plants and LHT1‐overexpressing plants. Slopes of regressions between accumulation of ¹³C‐labelled carbon and ¹⁵N‐labelled N were higher for LHT1‐overexpressing plants than wild type plants, while plants lacking expression of LHT1 did not display a significant regression between the two isotopes. Uptake of labelled organic N from soil tallied with that of labelled ammonium for wild type plants and LHT1‐overexpressing plants but was significantly lower for plants lacking expression of LHT1. When grown on agricultural soil plants lacking expression of LHT1 had the lowest, and plants overexpressing LHT1 the highest C/N ratios and natural δ¹⁵N abundance suggesting their dependence on different N pools. Our data show that LHT1 expression is crucial for plant uptake of organic N from soil.     

<Emphasis Type="Italic">Le</Emphasis>RALF,a plant peptide that regulates root growth and development,specifically binds to 25 and 120 kDa cell surface membrane proteins of <Emphasis Type="Italic">Lycopersicon peruvianum</Emphasis>

A photoaffinity analog of tomato leaf RALF peptide (LeRALF), ¹²⁵I-azido-LeRALF, bound saturably to tomato suspension cultured cells in the dark in a classical receptor binding assay. Classical kinetic analyses revealed that the analog interacted with a single binding site on the surface of the cells with a K_D of 0.8×10^–9 M, typical of known peptide hormone–receptor interactions in both plants and animals. The ¹²⁵I-azido-LeRALF, when exposed to UVB light in the presence of the cells, strongly labeled only two proteins of 25 kDa and 120 kDa, with the 25 kDa protein being more strongly labeled than the 120 kDa protein. The cell-surface localization of the interaction was indicated, as suramin, a known inhibitor of peptide–receptor interactions, and native LeRALF peptide competed with ¹²⁵I-azido-LeRALF labeling of both proteins. Two biologically inactive LeRALF analogs were not competitors. Incubation of ¹²⁵I-azido-LeRALF with suspension cultured cells in the dark, where it was fully active, could subsequently be totally dissociated from cells by acid washes, indicating that it was interacting at the cell surface and was not internalized. The ¹²⁵I-azido-LeRALF-labeled 25 kDa and 120 kDa proteins could not be solubilized from cell membranes by methods that release peripheral proteins, indicating that they are integral membrane components. The cumulative kinetic and biochemical evidence strongly indicates that the two proteins may be components of a LeRALF receptor complex.     

Structure dissection of zebrafish progranulins identifies a well‐folded granulin/epithelin module protein with pro‐cell survival activities

The ancient and pluripotent progranulins contain multiple repeats of a cysteine‐rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four β‐hairpins zipped together by six inter‐hairpin disulfide bonds. Prevalence of this disulfide‐enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four progranulins in the zebrafish genome, progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely folded conformations, module AaE from progranulin A and AbB from progranulin B were found to fold into the protopypic 4‐hairpin structure along with disulfide formation. Module AaE has the most‐rigid three‐dimensional structure with all four β‐hairpins defined using high‐resolution (H–¹⁵N) NMR spectroscopy, including 492 inter‐proton nuclear Overhauser effects, 23 ³J(HN,H_α) coupling constants, 22 hydrogen bonds as well as 45 residual dipolar coupling constants. Three‐dimensional structure of AaE and the partially folded AbB re‐iterate the conformational stability of the N‐terminal stack of two beta‐hairpins and varying degrees of structural flexibility for the C‐terminal half of the 4‐hairpin global fold of the GEM repeat. A cell‐based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human progranulin. Finally, this work highlights the remaining challenges in structure‐activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analysis of oxidized peptide fragments of the C‐terminal redox center in thioredoxin reductases by NMR spectroscopy

Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight‐membered ring structure. This eight‐membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans – 9:91). This was determined by using the ‘γ‐gauche effect’, which makes use of both ¹H‐NMR and two‐dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T? conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Rapid,robotic, small‐scale protein production for NMR screening and structure determination

Three‐dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time‐consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi‐automated protocol for isotopically‐labeled protein production using the Maxwell‐16, a commercially available bench top robot, that allows for single‐step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different ¹⁵N‐labeled proteins, accelerating the validation process by more than 10‐fold. The yield from a single channel of the Maxwell‐16 is sufficient for acquisition of a high‐quality 2D ¹H‐¹⁵N‐HSQC spectrum using a 3‐mm sample cell and 5‐mm cryogenic NMR probe. Maxwell‐16 screening of a control group of proteins reproduced previous validation results from conventional small‐scale expression screening and large‐scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par‐3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U‐¹⁵N,¹³C] protein prepared using the Maxwell‐16. This novel semi‐automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale‐up steps.     

Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States

A new protocol is described for the isotope (¹⁵N and ¹³C,¹⁵N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. ¹H,¹³C and ¹⁵N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the C^γ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.     

HIGH‐RESOLUTION MAGIC ANGLE SPINNING NMR ANALYSIS OF WHOLE CELLS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE) AND COMPARISON WITH 13C‐NMR AND DISTORTIONLESS ENHANCEMENT BY POLARIZATION TRANSFER 13C‐NMR ANALYSIS OF LIPOPHILIC EXTRACTS1

Lipid composition in extracted samples of Chaetoceros muelleri Lemmermann was studied with ¹³C‐NMR and distortionless enhancement by polarization transfer (DEPT) ¹³C‐NMR, resulting in well‐resolved ¹³C‐NMR spectra with characteristic resonance signals from carboxylic, olefinic, glyceryl, methylene, and methyl groups. The application of a DEPT pulse sequence aided in the assignment of methylene and methine groups. Resonance signals were compared with literature references, and signal assignment included important unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic and also phospholipids and glycerols. Results from the extracted samples were used to assign resonance signals in a high‐resolution magic angle spinning (HR MAS) DEPT ¹³C spectrum from whole cells of C. muelleri. The NMR analysis on whole cells yielded equally good information on fatty acids and also revealed signals from carbohydrates and amino acids. Broad resonance signals and peak overlapping can be a problem in whole cell analysis, but we found that application of HR MAS gave a well‐resolved spectrum. The chemical shift of metabolites in an NMR spectrum depends on the actual environment of nuclei during analysis, and some differences could therefore be expected between extracted and whole cell samples. The shift differences were small, and assignment from analysis of lipophilic extract could be used to identify peaks in the whole cell spectrum. HR MAS ¹³C‐NMR therefore offers a possibility for broad‐range metabolic profiling directly on whole cells, simultaneously detecting metabolites that are otherwise not detected in the same analytical set up and avoiding tedious extraction procedures.     

Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial ¹⁵N‐, ¹³C^α‐, and ¹³C'‐selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell‐free expression system, a scheme that involves ¹⁵N, 1‐¹³C, 2‐¹³C, fully ¹⁵N/¹³C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time‐shared triple‐resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non‐proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide ¹H and ¹⁵N resonances need to be obtained, even in cases where sensitivity is the limiting factor.