1-<Superscript>13</Superscript>C amino acid selective labeling in a <Superscript>2</Superscript>H<Superscript>15</Superscript>N background for NMR studies of large proteins

Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated ¹⁴N amino acids that are ¹³C labeled at the carbonyl position to a medium for uniform deuteration and ¹⁵N labeling. This has three important benefits over conventional ¹⁵N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the ¹³C labeled amino acid is very sharp since its alpha position is deuterated, (2) the ¹³C label at the carbonyl position is less prone to scrambling than the ¹⁵N at the α-amino position, and (3) the peaks for the 1-¹³C labeled amino acids can be identified easily from the large intensity reduction in the ¹H-¹⁵N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

NMR resonance assignments for sparsely <Superscript>15</Superscript>N labeled proteins

For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform ¹³C, ¹⁵N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected ¹⁵N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform ¹⁵N, ¹³C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach applicable to a single sample prepared with sparse ¹⁵N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein, Galectin-3. Limitations and future applications are discussed.     

Direct amide <Superscript>15</Superscript>N to <Superscript>13</Superscript>C transfers for solid-state assignment experiments in deuterated proteins

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only ¹H–¹⁵N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than ¹H–¹³C CP transfers, and employs a dipolar version of the INEPT experiment for N–C transfer. By avoiding H^N–C (H^N stands for amide protons) and C–C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired ¹³C spins.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576.     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

Measurement of the <Superscript>15</Superscript>N/<Superscript>14</Superscript>N ratio of phenylalanine in fermentation matrix by isotope ratio mass spectrometry

Objectives

To determine the origin of ¹⁵N-labeled phenylalanine in microbial metabolic flux analysis using ¹⁵N as a tracer, a method for measuring phenylalanine δ¹⁵N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.

Results

The original source of the ¹⁵N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a ¹⁵N-labeled inorganic nitrogen source, rather than ¹⁵N-labeled amino acids, was explored using this method.

Conclusions

The method described here can also be applied to the analysis of metabolic flux.

     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.     

<Superscript>15</Superscript>N/<Superscript>14</Superscript>N ratios of amino acids as a tool for studying terrestrial food webs: a case study of terrestrial insects (bees,wasps, and hornets)

Compound-specific stable isotope analysis (CSIA) of amino acids is a new method that enables estimates of trophic position for consumers in food webs. We examined the nitrogen isotopic composition (δ¹⁵N) of amino acids of Japanese social insects (three bee, three wasp, and four hornet species) to evaluate the potential of CSIA of amino acids in studies of terrestrial food webs. For wasps, we also examined samples at different growth stages (ranging from egg to adult) to assess the effect of metamorphosis on CSIA estimates of trophic position. The δ¹⁵N values of bulk tissues for Japanese social insects are only weakly correlated with the biologically expected trophic positions. In contrast, the trophic positions estimated from the δ¹⁵N values of amino acids (yielding values of between 2.0 and 2.3 for bees, between 2.8 and 3.3 for wasps, and between 3.5 and 4.1 for hornets) are consistent with the biologically expected trophic positions for these insects (i.e., 2.0 for bees, 3.0 for wasps, and 3.0–4.0 for hornets). Although large variability is observed among the δ¹⁵N values of individual amino acids (e.g., ranging from 3.0 to 14.9‰ for phenylalanine), no significant change is observed in the trophic position during wasp metamorphosis. Thus, the CSIA of amino acids is a powerful tool for investigating not only aquatic food webs but also terrestrial food webs with predatory insects.     

Protein <Superscript>19</Superscript>F-labeling using transglutaminase for the NMR study of intermolecular interactions

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic ¹⁵N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. ¹⁹F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, ¹⁹F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic ¹⁹F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The ¹⁹F-labeling method was 3.5-fold more sensitive than ¹⁵N-labeling, and could be combined with other chemical modification techniques such as lysine ¹³C-methylation. ¹³C-dimethylated-¹⁹F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of a calcium-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization.     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.     

Speeding up direct <Superscript>15</Superscript>N detection: hCaN 2D NMR experiment

Experiments detecting low gyromagnetic nuclei have recently been proposed to utilize the relatively slow relaxation properties of these nuclei in comparison to ¹H. Here we present a new type of ¹⁵N direct-detection experiment. Like the previously proposed CaN experiment (Takeuchi et al. in J Biomol NMR 47:271–282, 2010), the hCaN experiment described here sequentially connects amide ¹⁵N resonances, but utilizes the initial high polarization and the faster recovery of the ¹H nucleus to shorten the recycling delay. This allows recording 2D ¹⁵N-detected NMR experiments on proteins within a few hours, while still obtaining superior resolution for ¹³C and ¹⁵N, establishing sequential assignments through prolines, and at conditions where amide protons exchange rapidly. The experiments are demonstrated on various biomolecules, including the small globular protein GB1, the 22 kDa HEAT2 domain of eIF4G, and an unstructured polypeptide fragment of NFAT1, which contains many SerPro sequence repeats.     

Isotopic signature (<Superscript>15</Superscript>N/<Superscript>14</Superscript>N and <Superscript>13</Superscript>C/<Superscript>12</Superscript>C) confirms similarity of trophic niches of millipedes (Myriapoda,diplopoda) in a temperate deciduous forest

The species composition, abundance, and isotopic signature of millipedes (Myriapoda, Diplopoda) were investigated in seven biotopes of Kaluzhskie Zaseki State Nature Reserve. Nine Diplopoda species were found in total, and the local species diversity (within a sampling plot) reached seven species. The Diplopoda tissues were similar to the plant litter in the isotopic composition of nitrogen (δ¹⁵N was by 0.4‰ higher, on average), but were strongly enriched in heavy carbon (δ¹³C was by 4‰ higher, on average). Removal of mineral carbon from the cuticle reduced δ¹³C of Diplopoda by about 1.4‰ on average. Differences in the δ¹⁵N and δ¹³C values between the species did not exceed 2.5‰. Differences in the isotopic compositions of the considered species were small, and, it is impossible to distinguish particular trophic guilds in the Diplopoda community. Analysis of the published data confirmed that isotopic differentiation of millipedes was much less pronounced than in other investigated groups of soil animals. Hence, millipedes of the deciduous forest form a uniform trophic group.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of human osteopontin

Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin.