Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Stereospecific assignment of the asparagine and glutamine sidechain amide protons in proteins from chemical shift analysis

Side chain amide protons of asparagine and glutamine residues in random-coil peptides are characterized by large chemical shift differences and can be stereospecifically assigned on the basis of their chemical shift values only. The bimodal chemical shift distributions stored in the biological magnetic resonance data bank (BMRB) do not allow such an assignment. However, an analysis of the BMRB shows, that a substantial part of all stored stereospecific assignments is not correct. We show here that in most cases stereospecific assignment can also be done for folded proteins using an unbiased artificial chemical shift data base (UACSB). For a separation of the chemical shifts of the two amide resonance lines with differences ≥0.40 ppm for asparagine and differences ≥0.42 ppm for glutamine, the downfield shifted resonance lines can be assigned to H^δ21 and H^ε21, respectively, at a confidence level >95%. A classifier derived from UASCB can also be used to correct the BMRB data. The program tool AssignmentChecker implemented in AUREMOL calculates the Bayesian probability for a given stereospecific assignment and automatically corrects the assignments for a given list of chemical shifts.     

Conformational folding of xyloglucan side chains in aqueous solution from molecular dynamics simulation

Molecular dynamics simulation was carried out on xyloglucan with explicit water molecules to investigate the folding mechanism of side chains onto a main chain in aqueous solution. The model xyloglucan was composed of 12 beta-D-glucopyranoses as a main chain substituted with six galactoses and three xyloses as side chains. Two conditions were set for the ribbon-like main chain; one is restricted to be 'flat' and the other is without restriction. The free main chain of xyloglucan has a 'twisted' conformation as the major one. Conformational folding of side chains onto the main chain was analyzed with dihedral angles at each glycosidic linkage. In a 5-ns calculation, the xyloglucan has a tendency to contract in both the restricted and the free systems, but the mode of contraction is different. Side chains tend to stick onto the flat surface of the main chain in the restricted system, while they do not tightly do so in the free one; instead the main chain takes a twisted and sometimes embowed conformation. This result indicates that the main chain has greater attractive forces to bind side chains when it is flat, while it loses the ability as it is twisted.     

3d Hcch-Cosy-Tocsy experiment for the assignment of ribose and amino acid side chains in 13C labeled RNA and protein

A new 3D HCCH-COSY-TOCSY experiment is presented for the assignment of RNA sugar and protein side chains. The experiment, which combines COSY and TOCSY units, is more powerful than the sum of individual HCCH-COSY and HCCH-TOCSY pulse sequences. The experiment was applied to a 13C, 15N-labeled 26 mer RNA complexed with the antibiotic tobramycin, and a 12 kDa 13C, 15N-labeled FKBP12 protein sample. The power of HCCH-COSY-TOCSY is demonstrated through complete spin system assignments of sugars in the 26 mer RNA sample, which could not be assigned using a combination of HCCH-COSY, HCCH-TOCSY and 13C-edited NOESY experiments.     

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide ¹H and ¹⁵N resonances of amino acids. Frequency labeling of ¹³CO spins occurs during a period required for the ¹³C-¹⁵N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, ¹³C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.     

Improved NMR experiments with 13C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C^aliC^aro)H-TOCSY, is an HCCH-TOCSY in which all CH_n moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C^all-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C–¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for NH2 group rotation in buried asparagine side chains

Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.     

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

Summary It is demonstrated that sequential resonance assignment of the backbone ¹H and ¹⁵N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate ¹H with the intraresidue ¹⁵N and with the ¹⁵N resonance of the next residue. The experiments are preferably conducted in D₂O, where very high resolution in the ¹⁵N dimension can be achieved by using ²H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in ¹³C and ¹⁵N. Complete backbone and ¹³C/¹H resonance assignments are presented.     

Roles of the histidine and tryptophan side chains in the M2 proton channel from influenza A virus

The M2 protein form influenza A virus forms a tetrameric ion channel, which enables proton passage across biological membranes when the N-terminal side is acidified. Among the amino acid residues in the transmembrane domain of the M2 protein, His37 and Trp41 are essential for the pH-regulated proton conductance. Current knowledge about the structures and interactions of His37 and Trp41 suggests a model for the M2 ion channel, in which the channel is closed by a network of His37 hydrogen bonds at neutral pH and is opened by a His37-Trp41 cation-pi interaction at acidic pH.     

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

A simple and sensitive experiment for measurement of JCC couplings between backbone carbonyl and methyl carbons in isotopically enriched proteins

Summary A simple 2D difference experiment is described that allows quantitative measurement of ¹³C–¹³C J couplings between backbone carbonyl and side-chain carbons. Precise ³J_CC values were measured from data recorded in just 2 h for a 1-mM solution of the 20-kD complex between the protein calmodulin and a 26-residue synthetic peptide. The J couplings aid in determining the ¹ angles of valine, isoleucine and threonine residues, and in making stereospecific assignments of the Val C methyl groups. Error analysis indicates that the uncertainty in the derived J couplings is generally less than ca. 0.3 Hz.     

Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: a SDSL-EPR and MD study

Five singly spin labeled side chains at surface sites in the C-terminal domain of RGL2 protein have been analyzed to investigate the general relationship between nitroxide side chain mobility and protein structure. At these sites, the structural perturbation produced by replacement of a native residue with a nitroxide side chain appears to be very slight at the level of the backbone fold. The primary determinants of the nitroxide side chain mobility are backbone dynamics and tertiary interactions. On the exposed surfaces of alpha-helices, the side chain mobility is not restricted by tertiary interactions but appears to be determined by backbone dynamics, while in loop sites, the side chain mobility is even higher. For a better understanding of the changes in the EPR spectral line shape, molecular dynamics simulations were performed and found in agreement with EPR spectral data.     

BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in <Superscript>13</Superscript>C/<Superscript>15</Superscript>N labeled proteins

Aromatic amino-acid side chains are essential components for the structure and function of proteins. We present herein a set of NMR experiments for time-efficient resonance assignment of histidine and tyrosine side chains in uniformly ¹³C/¹⁵N-labeled proteins. The use of band-selective ¹³C pulses allows to deal with linear chains of coupled spins, thus avoiding signal loss that occurs in branched spin systems during coherence transfer. Furthermore, our pulse schemes make use of longitudinal ¹H relaxation enhancement, Ernst-angle excitation, and simultaneous detection of ¹H and ¹³C steady-state polarization to achieve significant signal enhancements.     

HNHC: a triple resonance experiment for correlating the H2, N1(N3) and C2 resonances in adenine nucleobases of <Superscript>13</Superscript>C-, <Superscript>15</Superscript>N-labeled RNA oligonucleotides

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in adenine nucleobases of ¹³C, ¹⁵N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a ²J-¹⁵N-HSQC and a ¹J-¹³C-HSQC and utilizes large ²J(H2, N1(N3)) and ¹J(H2, C2) couplings. The experiment was applied to a medium-size ¹³C, ¹⁵N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the ¹H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR 20:173–176 2001) is provided. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel NMR experiment for the sequential assignment of proline residues and proline stretches in 13C/15N-labeled proteins

A new pulse sequence is described for the sequential assignment of proline residues in 13C/15N-labeled proteins by correlating C and C chemical shifts of proline residues with the H chemical shift of the preceding residue. Notably, the experiment can provide the sequential connectivities in poly-proline stretches, which cannot be determined using standard triple resonance experiments. Excellent solvent suppression is achieved by coherence selection via a heteronuclear gradient echo. The new pulse sequence has been successfully applied to the 11 kDa HRDC domain.     

Sequential assignment of the 1H and 31P resonances of the double stranded deoxynucleotide d (ATGCAT)2 by 2D-NMR correlation spectroscopy

31p-1H and 1H-1H chemical shift correlation spectroscopy are jointly used for providing a complete assignment of sugar proton (except H5' and H5") and phosphorus resonances in the double stranded oligonucleotide d (ATGCAT)2. In contrast to previous methods the specific assignment of overcrowded H5' H5" proton resonances is not required. Using the H3'-P coupling and also the long range H4'-P coupling, this quite general method can be easily implemented on intermediate field spectrometer. The present results pave the way to the 1H and 31P resonance assignment of longer double-stranded oligonucleotides.     

An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in <Superscript>15</Superscript>N, <Superscript>13</Superscript>C labeled proteins

An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely ¹³C′(i), ¹⁵N(i), ¹H^N(i) connectivities in uniformly ¹⁵N/¹³C-labeled proteins. In comparison to the “out-and-back” style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the ¹³C′ dimension and on average 1.6 times higher sensitivity especially for residues in α-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16.     

Isozyme analysis of primary trisomies in beet (Beta vulgaris L.). Genetical characterization and techniques for chromosomal assignment of two enzyme-coding loci: leucine aminopeptidase and glutamate oxaloacetate transaminase

Segregating families of beet (Beta vulgaris) were used to verify the monofactorial inheritance of two enzyme-coding loci, leucine aminopeptidase (Lap1) and glutamate oxaloacetate transaminase (Got3). With a series of primary trisomies and using three methods to discriminate between the critical trisomic (the locus is situated on the triplicated chromosome) and the non-critical ones, it was possible to allocate the two loci to beet chromosomes I and II, respectively. For the locus Lap1 distorted segregation ratios were estimated, and the incorporation of three alleles into one plant was attempted. In the case of Got3 the measurement of the allele dosage effect after electrophoresis was chosen as the major strategy. The output of laser densitometric scans were subjected to the non-parametrical Wilcoxon-Mann-Whitney test.