SAILing toward larger protein structures

With a new method for optimizing the amino acid isotopic labeling pattern, stereo-array isotope labeling (SAIL), structures of large proteins (up to 50 kDa) can now be solved with ease with nuclear magnetic resonance (NMR) spectroscopy.     

Secoscabronine M, a novel diterpenoid from the Chinese bitter mushroom Sarcodon scabrosus

Secoscabronine M (1) is a hemiacetal cyathane diterpenoid that was isolated from the fruiting bodies of the basidiomycete Sarcodon scabrosus (Fr.) Karst. Compound 1 possesses a novel structure with a bond cleavage between C-3 and C-4. The structure of the new compound was elucidated by means of spectroscopic methods, including two-dimensional nuclear magnetic resonance experiments. The absolute configuration of 1 was established by analysis of circular dichroism spectroscopy and also by employing time-dependent density functional theory calculations. In addition, compound 1 was confirmed to be an equilibrium mixture of two epimers (15S and 15R) at position C-15 in polar solvents by one-dimensional nuclear magnetic resonance analysis.     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing ¹⁵N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a ¹⁵N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.     

Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution.

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.     

Qualitative aspects of hydrogen-deuterium exchange in the 1H, 13C, and 15N nuclear magnetic resonance spectra of viomycin in aqueous solution.

The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.     

Itaconate biosynthesis in Aspergillus terreus.

Itaconate biosynthesis was studied in intact cells of high-yield (RC4') and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism. Itaconate formation in RC4' required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using 14C- and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A. terreus is proposed.     

In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics

Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.     

Discovery of ligands for Nurr1 by combined use of NMR screening with different isotopic and spin-labeling strategies

A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR (19)F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (K(d) = 20 microM) for Nurr1 and identification of the binding site location through residue-specific (15)N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-(13)C.     

NMR observation of selected segments in a larger protein: central-segment isotope labeling through intein-mediated ligation

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.     

Non-invasive histochemistry of plant materials by magnetic resonance microscopy

Summary We have combined nuclear magnetic resonance (NMR) imaging on the microscopic scale with chemical shift selection to demonstrate the application of magnetic resonance imaging (MRI) to plant histochemistry. As an example of the method we have obtained separate images of the distribution of reserve oil and anethole in dried fennel mericarps. The technique can be employed to separately image the distribution of aromatics, carbohydrates, oils, water and possibly fatty acids in suitable plant materials.Abbreviations NMR nuclear magnetic resonance - MRI magnetic resonance imaging - COSY correlation spectroscopy - TMS tetramethylsilane     

NMR-derived folate-bound structure of dihydrofolate reductase 1 from the halophile Haloferax volcanii

Folate binds to dihydrofolate reductase (DHFR) to form a binary complex whose structure maintains the overall configuration of the enzyme; however, some significant changes are evident when a comparison is made to the enzyme. The structure of DHFR1 from the halophilic Halopherax volcanii was solved in its folate-bound form using nuclear magnetic resonance spectroscopy. NOE data obtained from the (15)N-NOESY-HSQC and (13)C-NOESY-HSQC experiments of the triply labeled ((1)H, (13)C, and (15)N) binary complex were used as input for the structure calculation with the Crystallography and Nuclear Magnetic Resonance System program. The resulting family of structures was compared with the enzyme solved by both nuclear magnetic resonance and X-ray crystallography and also to the mesophilic folate-bound enzyme from Escherichia coli. The binary complex exhibited less convergence of structure in the alpha2-helix and differences in the hinge residues D39 and A94. In comparison to the previously reported mesophilic binary complex solved by X-ray crystallography, the halophilic binary complex reported here does not agree with the convergence of the M20 loop to a single structure. The corresponding L21 loop of the halophilic binary complex family of structures solved by nuclear magnetic resonance indicates variability in this region.     

Isotope alteration caused by changes in biochemical composition of sedimentary organic matter

Stable carbon (C) and nitrogen (N) isotope ratios of sedimentary organic matter (OM) can reflect the biogeochemical history of aquatic ecosystems. However, diagenetic processes in sediments may alter isotope records of OM via microbial activity and preferential degradation of isotopically distinct organic components. This study investigated the isotope alteration caused by preferential degradation in surface sediments sampled from a eutrophic reservoir in Germany. Sediments were treated sequentially with hot water extraction, hydrochloric acid hydrolysis, hydrogen peroxide oxidation and di-sodium peroxodisulfate oxidation to chemically simulate preferential degradation pathways of sedimentary OM. Residue and extracts from each extraction step were analyzed using elemental analyzer-isotope ratio mass spectrometry and solid-state ¹³C nuclear magnetic resonance spectroscopy. Our results show that stable C and N isotope ratios reacted differently to changes in the biochemical composition of sedimentary OM. Preferential degradation of proteins and carbohydrates resulted in a 1.2‰ depletion of ¹³C, while the isotope composition of ¹⁵N remained nearly the same. Sedimentary δ¹⁵N values were notably altered when lignins and lipids were oxidized from residual sediments. Throughout the sequential fractionation procedure, δ¹³C was linearly correlated with the C:N of residual sediments. This finding demonstrates that changes in biochemical composition caused by preferential degradation altered δ¹³C values of sedimentary OM, while this trend was not observed for δ¹⁵N values. Our study identifies the influence of preferential degradation on stable C isotope ratios and provide additional insight into the isotope alteration caused by post-depositional processes.

     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell–cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1^C-ter) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1^1–186) was complementary to the most stable C-terminal fragments (Sic1^Δ186). Both Sic1^1–186 and Sic1^C-ter fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.     

Three-dimensional structure in solution of barwin, a protein from barley seed.

The solution structure of a 125-residue basic protein, barwin, has been determined using 1H nuclear magnetic resonance spectroscopy. This protein is closely related to domains in proteins encoded by wound-induced genes in plants. Analysis of the 1H nuclear Overhauser spectrum revealed the assignment of more than 1400 nuclear Overhauser effects. Twenty structures were calculated based on 676 nontrivial distance restraints, 152 torsion angle restraints (92 phi, 56 chi 1, and 4 omega for proline), and stereospecific assignments of 38 chiral centers, using distance geometry, simulated annealing, and restrained energy minimization. None of the distance restraints was violated by more than 0.5 A in any of the 20 structures, and none of the torsion angle restraints was violated by more than 1 degree in any of the structures. The RMS difference between the calculated and target interproton distance restraints is 0.033 A, and the average atomic RMS differences between the 20 structures and their geometric average are 1.23 A for backbone atoms and 1.73 A for all heavy atoms. The dominating structural feature of the protein is a well-defined four-stranded antiparallel beta-sheet, two parallel beta-sheets packed antiparallel to each other and four short alpha-helices. The binding site of barwin to the tetramer N-acetylglucosamine has been qualitatively investigated, and the dissociation constant of the complex has been determined using one-dimensional 1H nuclear magnetic resonance spectroscopy.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

Comparison of the potentiometric, (31)P NMR, and zero-point titration methods of determining ionized magnesium in erythrocytes

A simple and rapid method of determining ionized magnesium in erythrocytes using a potentiometric clinical analyzer, Microlyte 6 (Kone, Finland), was investigated. The erythrocyte cell membranes were destroyed using ultrasound. The results were compared with those obtained with the (31)P nuclear magnetic resonance spectroscopy method and the zero-point titration method using atomic absorption spectrometry. The results obtained from potentiometry and from the other methods did not differ significantly (Student t test, alpha = 0.01). Total magnesium concentration was determined using atomic absorption spectrometry.     

Solution conformation of endothelin-3 by H NMR and distance geometry calculations

Endothelin-3 dissolved in 10% aqueous acetic acid was studied by nuclear magnetic resonance spectroscopy. A total of 363 distances (143 intra-residue, 108 sequential and 112 long range) was compiled from the nuclear Overhauser effect spectra and used in distance geometry calculations. The molecule assumes a compact conformation stabilized by hydrophobic interactions of the side chains. There is a helix-like structure between the residues 9–15 and an extended strand at the N-terminus. The C-terminus is in close proximity to the bicyclic ring.