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

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

Simple and Rapid (Extraction) Protocol for NMR Metabolomics-Direct Measurement of Hydrophilic and Hydrophobic Metabolites Using Slice Selection.

Investigating the metabolic profiles of solid sample materials with solution nuclear magnetic resonance (NMR) spectroscopy requires the extraction of these metabolites. This is commonly done by using two immiscible solvents such as water and chloroform for extraction. Subsequent solvent removal makes these extraction procedures very time-consuming. To shorten the preparation time of the NMR sample, the following protocol is proposed: the metabolites from a solid or liquid sample are extracted directly in the NMR tube, the NMR tube is centrifuged, and the metabolite profiles in the aqueous and organic phases are determined by using slice-selective proton NMR experiments. This protocol was tested with 11 black teas and 11 green teas, which can be easily distinguished by their metabolic profiles in the aqueous phase. As a test case for liquid samples, 29 milk samples were investigated. The geographical origin of the diaries where the milk was processed could not be determined unequivocally from the metabolic profiles of the hydrophilic metabolites; however, this was easily seen in the lipid profiles. As shown for the different test samples, the extraction protocol in combination with slice-selection NMR experiments is suitable for metabolic investigations. Because samples are rapidly processed, this approach can be used to explore different extraction strategies for metabolite isolation.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

Quantitative analysis of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells using UHPLC-MS/MS.

γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer in HeLa cells using ultra-high-performance liquid chromatography-tandem mass spectrometry. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells.

Threonine 454 phosphorylation in Grainyhead-like 3 is important for its function and regulation by the p38 MAPK pathway.

The mammalian Grainyhead-like 3 (GRHL3) transcription factor is essential for epithelial development and plays a protective role against squamous cell carcinoma of the skin and of the oral cavity. A single nucleotide polymorphism (SNP) in GRHL3, rs141193530 (p.P455A), is associated with non-melanoma skin cancer in human patients. Moreover, it is known that this SNP, as well as another variant, rs41268753 (p.T454M), are associated with nonsyndromic cleft palate and that rs41268753 negatively affects GRHL3 transcriptional activity. These SNPs are located in adjacent codons of the GRHL3 gene, and the occurrence of either SNP abolishes a putative threonine-proline phosphorylation motif at T454 in the encoded protein. The role of phosphorylation in regulating mammalian GRHL function is currently unknown. In this work we show that GRHL3 is phosphorylated at several residues in a human keratinocyte cell line, among them at T454. This site is essential for the full transcriptional activity of GRHL3. The T454 residue is phosphorylated by p38 MAPK in vitro and activation of p38 signaling in cells causes an increase in GRHL3 activity. The regulation of GRHL3 function by this pathway is dependent on T454, as the substitution of T454 with methionine inhibits the activation of GRHL3. Taken together, our results show that T454 is one of the phosphorylated residues in GRHL3 in keratinocytes and this residue is important for the upregulation of GRHL3 transcriptional activity by the p38 pathway.

Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions.

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Hydrazide functionalized magnetic nanoparticles for specific extraction of N-terminal serine and threonine peptides.

In this study, we present hydrazide functionalized magnetic nanoparticles as a sorbent prepared by a new and facile method. Scanning electron microscope and Fourier transform infrared were used for characterizing the synthesized nanoparticles. The ability of the sorbent to extract N-terminal serine and threonine peptides was evaluated. The peptides were modified by oxidation of the hydroxyl group in the 1,2-amino alcohol structure before extraction. These aldehyde-forms of peptides were specifically bonded to the hydrazide groups of the sorbent. The formed hydrazone bonds were cleaved in the presence of hydroxylamine reagent. Finally, the oximated peptides were released and quantified with a high-performance liquid chromatography-diode array spectroscopy. The effects of experimental parameters including extraction time, elution time and elution volume on extraction efficiency were also investigated. The required time for the extraction process to reach equilibrium and elution time was only 8 h. The adsorption efficiency of the sorbent was 79 and 77% for peptides with N-terminal serine and threonine, respectively. The sorbent showed good specificity for extracting the peptides. In addition, the extraction efficiency of the sorbent remained constant in the presence of a non-N-terminal serine and threonine peptide as interference.

Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe, Lys:Thr, Lys:His, and Lys:Val alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription.

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of ß-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation.

Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

O-GlcNAcylation of amyloid-ß precursor protein at threonine 576 residue regulates trafficking and processing.

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-ß (Aß) derived from proteolytic processing of amyloid-ß precursor protein (APP). APP undergoes post-translational modification including N- and O-glycosylation. O-GlcNAcylation is a novel type of O-glycosylation, mediated by O-GlcNAc transferase attaching O-ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of the target proteins. O-GlcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GlcNAcylated APP using the O-GlcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aß production. However, O-GlcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GlcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GlcNAcylation levels, indicating that these sites were endogenously O-GlcNAcylated. Thr 576 was the major O-GlcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and Aß production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GlcNAcylation site in APP and indicate that O-GlcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GlcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics.

The dietary protein paradox and threonine 15 N-depletion: Pyridoxal-5'-phosphate enzyme activity as a mechanism for the δ15 N trophic level effect.

RATIONALE: Nitrogen stable isotope ratios (δ15 N values) are used to reconstruct dietary patterns, but the biochemical mechanism(s) responsible for the diet to tissue trophic level effect and its variability are not fully understood. Here δ15 N amino acid (AA) values and physiological measurements (nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates) are used to investigate increased dietary protein consumption and oxidative stress (vitamin E deficiency) in rat total plasma protein. METHODS: Using gas chromatography/combustion/isotope ratio mass spectrometry, the δ15 N values from N-pivaloyl-i-propyl esters of 15 AAs are reported for rats (n = 40) fed casein-based diets with: adequate protein (AP, 13.8%; n = 10), medium protein (MP, 25.7%; n = 10), high protein (HP, 51.3%; n = 10) or HP without vitamin E (HP-E; n = 10) for 18 weeks. RESULTS: Between the HP and AP groups, the δ15 NAA values of threonine (-4.0‰), serine (+1.4‰) and glycine (+1.2‰) display the largest differences and show significant correlations with: nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates. This indicates increased AA catabolism by the dietary induction of shared common metabolic pathways involving the enzymes threonine ammonia-lyase (EC 4.3.1.19), serine hydroxymethyltransferase (EC 2.1.2.1) and the glycine cleavage system (EC 2.1.2.10). The δ15 NAA values of the HP-E and HP groups were not found to be significantly different. CONCLUSIONS: The 15 N-depleted results of threonine are linked to increased activity of threonine ammonia-lyase, and show potential as a possible biomarker for protein intake and/or gluconeogenesis. We hypothesize that the inverse nitrogen equilibrium isotope effects of Schiff base formation, between AAs and pyridoxal-5'-phosphate cofactor enzymes, play a key role in the bioaccumulation and depletion of 15 N in the biomolecules of living organisms and contributes to the variability in the nitrogen trophic level effect. Copyright © 2017 John Wiley & Sons, Ltd.

Crystallisation from a Water-in-Oil Emulsion as a Route to Enantiomer Separation: The Case of DL-Threonine.

The use of crystalliation as a means of separating enantiomers is well known. The utility of commonly applied seeding approaches is limited by the ultimate crystallisation of the antipode. Here we demonstrate how the combination of colloid science and crystal chemistry can lead to an emulsion based process yielding robust separation of a purified solid and impure liquid phases with ultimate product ee of up to 90 %. Threonine is used as a model to demonstrate the viability of the method but it is clear that extension to include, for example, simultaneous racemisation within the disperse phase is easily possible and would transform this from a separation to a preparation process.

Threonine versus isothreonine in synthetic peptides analyzed by high-resolution liquid chromatography/tandem mass spectrometry.

RATIONALE: One of the problems in proteogenomic research aimed at identification of variant peptides is the presence of peptides with amino acid isomers of different origin in the analyzed samples. Among the most challenging examples are peptides with threonine and isothreonine (homoserine) in their sequences. Indeed, the latter residue may appear in vitro as a methionine substitution during sample preparation for shotgun proteome analysis. Yet, this substitution of Met to isoThr is not encoded genetically and should be unambiguously distinguished from, e.g., point mutations in proteins that result in Met conversion to Thr. METHODS: In this work we compared tandem mass (MS/MS) spectra produced by an Orbitrap mass spectrometer of Thr- and isoThr-containing tryptic peptides and found a distinctive feature in their collisionally activated fragmentation patterns. RESULTS: Up to 84% of MS/MS spectra for peptides containing isoThr residues have been positively specified. We also studied the differences in retention times for peptides containing Thr isoforms that can be further used for their distinction. CONCLUSIONS: Threonine can be distinguished from isothreonine by its retention time and HCD fragmentation pattern, specifically relative intensity of the bn - product ion, which can be further used in proteomic research. Copyright © 2016 John Wiley & Sons, Ltd.

Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly.

RATIONALE: The "Threonine Anomaly" relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in 15 N relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its 15 N depletion. Plots of delta 15 N against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in 15 N is clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright © 2016 John Wiley & Sons, Ltd.

Threonine Requirement of the Enterally Fed Term Infant in the First Month of Life.

OBJECTIVE: Threonine is one of the essential amino acids. Its major fate is incorporation into intestinal mucosal proteins and synthesis of secretory glycoproteins. Therefore, it has an important function in the neonatal gut barrier integrity. The objective was to quantify the threonine requirement in fully enterally fed term neonates by means of the indicator amino acid oxidation (IAAO) method, using L-[1-C]phenylalanine as indicator. METHODS: After a 24-hour test diet adaptation, containing randomly assigned amounts of threonine (range 5-182 mg · kg · day), the participating neonates received a primed continuous infusion of [C]bicarbonate and L-[1-C]phenylalanine. At baseline and during the plateau phase of both infusions, breath samples were obtained for CO2. The fractional L-[1-C]phenylalanine oxidation (FCO2) was estimated and plotted against the threonine intakes. Biphasic linear regression crossover analysis was used to calculate the breakpoint of the FCO2, representing the mean threonine requirement. Data are presented as mean ±â€ŠSD. RESULTS: Thirty-two term neonates (gestational age 39 ±â€Š1 weeks, birth weight 3.3 ±â€Š0.3 kg, mean postnatal age 10 ±â€Š4 days) were studied. The mean threonine requirement was estimated to be 68 mg · kg · day with an upper and lower 95% confidence interval of 104 and 32 mg · kg · day, respectively (r = 0.37). CONCLUSIONS: The determined threonine requirement is extremely close to the existing requirement recommendations (∼90% of the present World Health Organization requirement guidelines). Infant formula preparations presently on the market, however, contain up to twice as much threonine as recommended. The threonine intake in formula-fed infants may therefore be reduced considerably.

Free amino acids in stimulated and unstimulated whole saliva: advantages or disadvantages.

This study determines the mean concentrations of free amino acids in stimulated and unstimulated whole saliva in healthy young adults. Standardised salivary amino acids as a substitute for their counterpart in blood, searched for the source of free amino acids in saliva, the probable correlation between particular amino acids with caries experience. Stimulated and unstimulated whole saliva were collected by the draining method in 31 dental students. Saliva was purified, and amino acids were separated by high-performance liquid chromatography. DMFT scores were recorded, and the relation of amino acids to caries experience was explored by generalised linear model. Almost all amino acids had higher concentration in unstimulated whole saliva than in stimulated saliva. The normal range of amino acids (95% CI) and their natural logarithm were defined. There was a significant relationship between caries experience and threonine (P < 0·008), citrulline (P < 0·023) and ornithine (P < 0·001) as a detrimental factor, whereas serin (P < 0·026), glutamine (P < 0·015) and phenylalanine (P < 0·014) had an inhibiting effect on caries. However, in comparison, salivary flow rate (P < 0·013) was a more preventive factor than amino acids. Amino acids in saliva contribute as a marker, instead of their counterpart in blood. Unstimulated saliva had higher concentration of amino acids. Amino acids have different impact on caries and may be one of underlying risk factors for caries experience.

Differential detergent extraction of mycobacterium marinum cell envelope proteins identifies an extensively modified threonine-rich outer membrane protein with channel activity.

A striking characteristic of mycobacteria is the presence of an unusual outer membrane which forms a thick permeability barrier and provides resistance to many antibiotics. Although specialized proteins must reside in this layer, only few mycolate outer membrane (MOM) proteins have been identified to date. Their discovery is complicated by difficulties in obtaining good separation of mycobacterial inner and outer membranes. During our efforts to identify novel mycobacterial outer membrane proteins (MOMPs), we discovered that we can enrich for MOMPs using differential solubilization of mycobacterial cell envelopes. Subsequently, these different fractions were analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This proteomic analysis confirmed that our marker proteins for inner membrane and MOM were found in their expected fractions and revealed a few interesting candidate MOMPs. A number of these putative MOMPs were further analyzed for their expression and localization in the cell envelope. One identified MOMP, MMAR_0617 of Mycobacterium marinum, was purified and demonstrated to form a large oligomeric complex. Importantly, this protein showed a clear single-channel conductance of 0.8 ± 0.1 ns upon reconstitution into artificial planar lipid bilayers. The most surprising feature of MMAR_0617 is a long C-terminal threonine-rich domain with extensive modifications. In summary, we have identified a novel mycobacterial outer membrane porin with unusual properties.

Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

Nuclear spin singlet states as a contrast mechanism for NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectra of complex chemical mixtures often contain unresolved or hidden spectral components, especially when strong background signals overlap weaker peaks. In this article we demonstrate a quantum filter utilizing nuclear spin singlet states, which allows undesired NMR spectral background to be removed and target spectral peaks to be uncovered. The quantum filter is implemented by creating a nuclear spin singlet state with spin quantum numbers j = 0, mz = 0 in a target molecule, applying a continuous RF field to both preserve the singlet state and saturate the magnetization of undesired molecules and then mapping the target molecule singlet state back into an NMR observable state so that its spectrum can be read out unambiguously. The preparation of the target singlet state can be carefully controlled with pulse sequence parameters, so that spectral contrast can be achieved between molecules with very similar structures. We name this NMR contrast mechanism 'Suppression of Undesired Chemicals using Contrast-Enhancing Singlet States' (SUCCESS) and we demonstrate it in vitro for three target molecules relevant to neuroscience: aspartate, threonine and glutamine.