Specific ion effects on macromolecular interactions in Escherichia coli extracts

Protein characterization in situ remains a major challenge for protein science. Here, the interactions of ΔTat‐GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). ΔTat‐GB1 was found to participate in high molecular weight complexes that remain intact at physiologically‐relevant ionic strength. This observation helps to explain why ΔTat‐GB1 was not detected by in‐cell NMR spectroscopy. Extracts pre‐treated with RNase A had a different SEC elution profile indicating that ΔTat‐GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of ΔTat‐GB1 could be disrupted by biologically‐relevant multivalent ions. The most effective shielding of interactions occurred in Mg²⁺‐containing buffers. Moreover, a combination of RNA digestion and Mg²⁺ greatly enhanced the NMR detection of ΔTat‐GB1 in cell extracts.     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Effects of carbonylcyanide-m-chlorophenylhydrazone (CCCP) and acetate on Escherichia coli O157:H7 and K-12: uncoupling versus anion accumulation

Non-growing cells of Escherichia coli O157:H7 and K-12 that were incubated anaerobically in sodium phosphate buffer at pH 6.5 consumed glucose at a rate of approximately 8 μmol·(mg protein)⁻¹·h⁻¹ and had intracellular pH values of 7.3 and 7.5, respectively. The uncoupler, carbonylcyanide-m-chlorophenylhydrazone (CCCP), caused a marked decrease in intracellular pH, ATP and potassium of both strains. Low concentrations of CCCP stimulated glucose consumption rate, but higher concentrations were inhibitory. Acetate also caused a decrease in intracellular pH, but it never caused a large decrease in glucose consumption rate. Acetate decreased the intracellular ATP of E. coli K-12, but it had no effect on the ATP of O157:H7. Acetate had no effect on the intracellular potassium of E. coli O157:H7, and acetate-treated K-12 cells had even more potassium than untreated controls. Based on these results, acetate and CCCP appear to have different effects on E. coli. The comparison of E. coli O157:H7 and K-12 indicated that intracellular pH, acetate accumulation and intracellular potassium were related. E. coli K-12 maintained a higher intracellular pH than O157:H7, accumulated more acetate and had a greater intracellular potassium.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> 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.     

Efficient condensed-phase production of perdeuterated soluble and membrane proteins

Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality ²H, ¹³C, ¹⁵N-enriched protein samples at 2.5–10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced ²H, ¹³C, ¹⁵N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40× condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce ²H, ¹³C, ¹⁵N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein’s structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10–40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.     

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.     

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.     

Application of Two‐Dimensional NMR Spectroscopy to Metabotyping Laboratory Escherichia coli Strains

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one‐dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two‐dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed ¹H,¹³C‐HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1‐Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D‐NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.     

Characterisation of cisplatin coordination sites in cellular <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry

Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl₂(NH₃)₂] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b⁺ and y⁺ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.     

The effects of high concentrations of ionic liquid on GB1 protein structure and dynamics probed by high-resolution magic-angle-spinning NMR spectroscopy

Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentrations of ionic liquids, has been challenging. In the present work the ¹³C, ¹⁵N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid–protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C₄-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6–3.5 M, which corresponds to 10–60% v/v). Interactions between GB1 and [C₄-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in ¹⁵N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C₄-mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of molecular mechanisms of ionic liquid–protein interactions.     

Production of isotopically labeled heterologous proteins in non-<Emphasis Type="Italic">E. coli</Emphasis> prokaryotic and eukaryotic cells

The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression systems are required for producing proteins for biomolecular NMR analyses. In this paper, we review the recent advances in expression systems for isotopically labeled heterologous proteins, utilizing non-E. coli prokaryotic and eukaryotic cells.     

In situ structural biology using in-cell NMR

BackgroundAccumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades.Scope of reviewIn this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed.Major conclusionsA number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli.General significanceIn-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro.     

1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I

Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled ¹³C/¹⁵N recombinant protein was produced in E. coli. Here we report the complete NMR ¹⁵N, ¹³C and ¹H chemical shifts assignments of Stn I at pH 4.0 and 25°C (BMRB No. 15927).     

Probing non-specific interactions of Ca2+-calmodulin in E. coli lysate

The biological environment in which a protein performs its function is a crowded milieu containing millions of molecules that can potentially lead to a great many transient, non-specific interactions. NMR spectroscopy is especially well suited to study these weak molecular contacts. Here, non-specific interactions between the Ca²⁺-bound form of calmodulin (CaM) and non-cognate proteins in Escherichia coli lysate are explored using Ile, Leu, Val and Met methyl probes. Changes in CaM methyl chemical shifts as a function of added E. coli lysate are measured to determine a minimum ‘average’ dissociation constant for interactions between Ca²⁺-CaM and E. coli lysate proteins. ²H R ₂ and ¹³C R ₁ spin relaxation rates report on the binding reaction as well. Our results further highlight the power of methyl containing side-chains for characterizing biomolecular interactions, even in complex in-cell like environments.     

Utilization of lysine 13C-methylation NMR for protein–protein interaction studies

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive ¹³C-methylation of the amino group of the lysine side-chain by ¹³C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. ¹³C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine ¹³C-methylation NMR to monitor protein–protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the ¹H–¹³C HSQC spectra of the ¹³C-methylated-lysine methyl groups. The lysine ¹³C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.     

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by ¹H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from ¹H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the ¹H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-of-saturation by cross-relaxation method.     

High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment

Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and ¹D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation.