Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins

Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with ¹⁵N or ¹³C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with ¹⁵N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells.     

SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [(13)C(3)(15)N]-pantothenate (vitamin B(5)), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2-3 weeks.     

Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly (¹³C/¹⁵N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled‐2 sulfatide‐binding motif

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea‐equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled‐2 (Dab2) sulfatide‐binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane‐binding, recombinant peptides that co‐purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S‐transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea‐equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at ?80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co‐purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment

Summary A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in ¹³C-/¹⁵N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.     

The efficient synthesis of isotopically labeled peptide-derived Amadori products and their characterization

Protein glycation is often a cause of diabetes-associated complications. The isotopically labeled peptide-derived Amadori products may serve as standards for quantitative determination of the glycated proteins. In this paper, we discussed various approaches to the synthesis of Amadori products labeled selectively with stable isotopes ²H, ¹³C and ¹⁸O.     

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.     

Overexpression of myoglobin and assignment of its amide,Cα and Cβ resonances

Summary Sperm whale apomyoglobin was expressed to high levels on minimal media and isotopically labeled with ¹³C and ¹⁵N nuclei. The isotopically labeled apoprotein was purified to homogeneity in a single step by reversed-phase chromatography and reconstituted with hemin and carbon monoxide gas for NMR analysis. Sequence-specific backbone ¹H^N, ¹⁵N and ¹³C as well as side-chain ¹³C resonance assignments have been made for over 90% of the amino acids in the carbon monoxide complex of the protein. Resonance assignments were made by analysis of a series of 3D triple resonance spectra measured on the uniformly labeled sample. These assignments will provide the basis for analyzing the effects of point site mutations on the structure, stability and dynamics of the protein in solution.     

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with ¹⁵N,¹³C by providing them with isotopically labeled bacteria. ²H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the “test” GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.     

A method for detecting protein-DNA interactions at sites of chromatin replication

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-³²P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.     

Large-scale total synthesis of <Superscript>13</Superscript>C<Subscript>3</Subscript>-labeled citrinin and its metabolite dihydrocitrinone

The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three ¹³C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.     

Sequential protein expression and selective labeling for in-cell NMR in human cells

BackgroundIn-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells.MethodsWe established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR.ResultsSOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by ¹H–¹⁵N heteronuclear NMR.Conclusions and general significanceWe showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at     

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H?2CO]⁺ ions derived from isoflavones and [M+H?B-ring?CO]⁺ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1′,2′,3′,4′,5′,6′,2,3,4-¹³C₉] and [2′,3′,5′,6′,2-D₅] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied ¹³C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H?2CO]⁺ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H?B-ring?CO]⁺ ion is demonstrated to contain a C₂H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSⁿ.