Structure of SAGA and mechanism of TBP deposition on gene promoters.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.

Yeast-based reference materials for quantitative metabolomics.

We introduce a new concept of yeast-derived biological matrix reference material for metabolomics research relying on in vivo synthesis of a defined biomass, standardized extraction followed by absolute quantification with isotope dilution. The yeast Pichia pastoris was grown using full control- and online monitoring fed-batch fermentations followed by fast cold methanol quenching and boiling ethanol extraction. Dried extracts served for the quantification campaign. A metabolite panel of the evolutionarily conserved primary metabolome (amino acids, nucleotides, organic acids, and metabolites of the central carbon metabolism) was absolutely quantified by isotope dilution utilizing uniformly labeled 13C-yeast-based internal standards. The study involved two independent laboratories employing complementary mass spectrometry platforms, namely hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Homogeneity, stability tests (on a panel of >70 metabolites over a period of 6 months), and excellent biological repeatability of independent fermentations over a period of 2 years showed the feasibility of producing biological reference materials on demand. The obtained control ranges proved to be fit for purpose as they were either superior or comparable to the established reference materials in the field.

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study.

BACKGROUND: Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. METHODS: A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K. RESULTS: In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol- 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K. CONCLUSIONS: Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.

Recent developments of methyl-labeling strategies in Pichia pastoris for NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryotic co-translational and post-translational modification, as well as high-density fermentation, Pichia pastoris has been a cost-effective platform strain for 13C, 15N-labeling and deuterium labeling since the early 2000's. Recently, some substantial progress has been made in methyl-labeling, such as the feasibility of 13C isoleucine δ1 methyl-labeling in perdeuterated background and the increased uptake of the Val/Leu precursor. Here, we systematically introduce the isotope-labeling strategies in P. pastoris, including strain engineering and detailed fermentation protocols in 13C, 15N-labeling and methyl-labeling, providing instructions and guidance for the future improvement of sample preparation for NMR spectroscopy.

Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system.

Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast.

BACKGROUND: Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. RESULTS: In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae, were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha. CONCLUSIONS: The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

Preparative supercritical fluid chromatography for lipid class fractionation-a novel strategy in high-resolution mass spectrometry based lipidomics.

In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level. Graphical Abstract.

Yeasts Influence Host Selection and Larval Fitness in Two Frugivorous Carpophilus Beetle Species.

We explored how gut-associated yeasts influence olfactory behaviour and resource use in two pest species of Carpophilus beetle that co-exist in Australian stone fruits. Molecular analysis of yeasts isolated from the gut of C. davidsoni (prefers ripe fruits) and C. hemipterus (prefers overripe and rotting fruits) revealed that the predominant species were Pichia kluyveri and Hanseniaspora guilliermondii. In olfactory attraction and oviposition trials, adult beetles preferred H. guilliermondii over P. kluyveri, and follow up GC-MS analysis revealed unambiguous differences between the odour profiles of these yeasts. In contrast to behavioural trials, larval feeding assays showed that fruit substrates inoculated with P. kluyveri yielded significantly faster development times, higher pupal mass, and a greater number of adult beetles, compared to H. guilliermondii - in other words, the lesser preferred yeast (by foraging adults) was more suitable for larval survival. Moreover, whilst larvae of both species survived to adulthood when fed solely on P. kluyveri (i.e. without a fruit substrate), only larvae of C. davidsoni could develop on H. guilliermondii; and only C. davidsoni reached adulthood feeding on a yeast-free fruit substrate. We discuss how these findings may relate to adaptations towards early colonising of fruits by C. davidsoni, enabling differences in resource use and potentially resource partitioning in the two beetles. More broadly, consideration of microbial interactions might help develop host selection theory. Our results could pave the way to more powerful attractants to mass-trap and monitor Carpophilus pests in fruit orchards.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris.

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

High-yield production of spider short-chain insecticidal neurotoxin Tx4(6-1) in Pichia pastoris and bioactivity assays in vivo.

Short-chain insecticidal neurotoxin Tx4(6-1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6-1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6-1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6-1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6-1) was purified from a 400 ml fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6-1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6-1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5-8.5 µg/g body weight. Dietary toxicity was not observed in silkworm larvae.

Proposing a validation scheme for 13C metabolite tracer studies in high-resolution mass spectrometry.

13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.

Accurate and cost-effective prediction of HBsAg titer in industrial scale fermentation process of recombinant Pichia pastoris by using neural network based soft sensor.

In the current work, the attempt was made to apply best-fitted artificial neural network (ANN) architecture and the respective training process for predicting final titer of hepatitis B surface antigen (HBsAg), produced intracellularly by recombinant Pichia pastoris Mut+ in the commercial scale. For this purpose, in large-scale fed-batch fermentation, using methanol for HBsAg induction and cell growth, three parameters of average specific growth rate, biomass yield, and dry biomass concentration-in the definite integral form with respect to fermentation time-were selected as input vectors; the final concentration of HBsAg was selected for the ANN output. Used dataset consists of 38 runs from previous batches; feed-forward ANN 3:5:1 with training algorithm of backpropagation based on a Bayesian regularization was trained and tested with a high degree of accuracy. Implementing the verified ANN for predicting the HBsAg titer of the five new fermentation runs, excluded from the dataset, in the full-scale production, the coefficient of regression and root-mean-square error were found to be 0.969299 and 2.716774, respectively. These results suggest that this verified soft sensor could be an excellent alternative for the current relatively expensive and time-intensive analytical techniques such as enzyme-linked immunosorbent assay in the biopharmaceutical industry.

Application of hydrocyclone for separation of Pichia pastoris produced r-HBsAg from fermentation culture: impact of concentration and pressure on hydrocyclone performance.

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated. Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R2 = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.

Integration of size-exclusion chromatography and ultracentrifugation for purification of recombinant hepatitis B surface antigen: An alternative method for immunoaffinity chromatography.

In purification process of recombinant hepatitis B surface antigen (rHBsAg), immunoaffinity chromatography (IAF) is one of the most important and effective steps in rHBsAg purification. However, the buffer composition and the interaction of ligands-rHBsAg often lead to disassembly, deformation, and clumping of a portion of these virus-like particles (VLPs). Besides, the expensive media, variable biospecific ligand density and the possibility of product contamination are other reported drawbacks of using IAF which makes the production process of rHBsAg more challenging. This study investigated the possibility of substituting IAF with purification methods of size-exclusion chromatography (SEC) and ultracentrifugation. In the SEC, the efficacy of rHBsAg purification was examined by four different media in which Toyopearl HW 65S resin demonstrated the best results. By integrating Toyopearl HW 65S resin - with a bed height of 51 cm - and ultracentrifugation process at 47,000 rpm for 48 hr, 95% of protein impurities were removed. Compared to the IAF in rHBsAg production, the purified sample contained a higher percentage of multimeric rHBsAg particles without any noticeable monomer and aggregate forms. The result of this study indicates that the proposed integrated system could be an efficient mild purification alternative for conventional IAF.

Functional regions of the peroxin Pex19 necessary for peroxisome biogenesis.

The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division, and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted the direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-containing PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10 while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reversed the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3- and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.

Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor.

Tryptophan indole 15N-1H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15N-1H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15N-1H resonances is challenging. Here we exploit the unique indole 15N-1H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A2AAR global protein fold and ligand binding activity are preserved in these A2AAR variants. The indole 15N-1H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15N-1H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the GαS intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H-13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.

On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies.

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

METLIN: A Technology Platform for Identifying Knowns and Unknowns.

METLIN originated as a database to characterize known metabolites and has since expanded into a technology platform for the identification of known and unknown metabolites and other chemical entities. Through this effort it has become a comprehensive resource containing over 1 million molecules including lipids, amino acids, carbohydrates, toxins, small peptides, and natural products, among other classes. METLIN's high-resolution tandem mass spectrometry (MS/MS) database, which plays a key role in the identification process, has data generated from both reference standards and their labeled stable isotope analogues, facilitated by METLIN-guided analysis of isotope-labeled microorganisms. The MS/MS data, coupled with the fragment similarity search function, expand the tool's capabilities into the identification of unknowns. Fragment similarity search is performed independent of the precursor mass, relying solely on the fragment ions to identify similar structures within the database. Stable isotope data also facilitate characterization by coupling the similarity search output with the isotopic m/ z shifts. Examples of both are demonstrated here with the characterization of four previously unknown metabolites. METLIN also now features in silico MS/MS data, which has been made possible through the creation of algorithms trained on METLIN's MS/MS data from both standards and their isotope analogues. With these informatic and experimental data features, METLIN is being designed to address the characterization of known and unknown molecules.