4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins

A four-dimensional (4D) APSY (automated projection spectroscopy)-HBCB(CG)CDHD experiment is presented. This 4D experiment correlates aromatic with aliphatic carbon and proton resonances from the same amino acid side chain of proteins in aqueous solution. It thus allows unambiguous sequence-specific assignment of aromatic amino acid ring signals based on backbone assignments. Compared to conventional 2D approaches, the inclusion of evolution periods on ¹H^β and ¹³C^δ efficiently removes overlaps, and provides two additional frequencies for consequent automated or manual matching. The experiment was successfully applied to three proteins with molecular weights from 6 to 13 kDa. For the complementation of the assignment of the aromatic resonances, TOCSY- or COSY-based versions of a 4D APSY-HCCH^aro sequence are proposed.     

Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

Several techniques for spectral editing of 2D ¹³C?C¹³C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N?CCO peaks through ¹³C?C¹⁵N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH₂) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other ??-pulse is shifted from the center of a rotor period t_r by about 0.15 t_r. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled ¹³C?C¹H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via ¹³C spin exchange. The efficiencies of these spectral editing techniques range from 60?% for the COO and dynamic selection experiments to 25?% for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.     

The use of heteronuclear cross-polarization for backbone assignment of 2H-, 15N- and 13C-labeled proteins: A pulse scheme for triple-resonance 4D correlation of sequential amide protons and 15N

Summary A new four-dimensional pulse scheme is described for the main-chain assignment of proteins by means of the J connectivity of the amide proton and nitrogen resonances of adjacent residues. Since the new experiment, 4D CP-HN(COCA)NH, involves heteronuclear cross-polarization for magnetization transfer from ¹³C=O to ¹⁵N via ¹³C, a relatively strong WALTZ-16 decoupling rf field is applied to ¹³C during magnetization transfer. Consequently, ¹³C is effectively decoupled from its attached ²H in the case of deuterated proteins, in the absence of a decoupling rf field for ²H. This efficiently improves the sensitivity of the experiment through ¹³C line narrowing. The experiment was performed on a randomly 60% deuterated protein, and the sensitivity of the final 4D spectrum was found to be excellent.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Methods for sequential resonance assignment in solid, uniformly 13C, 15N labelled peptides: quantification and application to antamanide

The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly ¹³C-¹⁵N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the C and aliphatic side-chain ¹³C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P9¹ ₁₂ TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the ¹³C and ¹⁵N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann–Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively.     

Anthranilic acid,the new player in the ensemble of aromatic residue labeling precursor compounds

The application of metabolic precursors for selective stable isotope labeling of aromatic residues in cell-based protein overexpression has already resulted in numerous NMR probes to study the structural and dynamic characteristics of proteins. With anthranilic acid, we present the structurally simplest precursor for exclusive tryptophan side chain labeling. A synthetic route to ¹³C, ²H isotopologues allows the installation of isolated ¹³C–¹H spin systems in the indole ring of tryptophan, representing a versatile tool to investigate side chain motion using relaxation-based experiments without the loss of magnetization due to strong ¹J_CC and weaker ²J_CH scalar couplings, as well as dipolar interactions with remote hydrogens. In this article, we want to introduce this novel precursor in the context of hitherto existing techniques of in vivo aromatic residue labeling.     

Detection of side-chain proton resonances of fully protonated biosolids in nano-litre volumes by magic angle spinning solid-state NMR

We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C–13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 µg using a MAS frequency of 70 kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Hα resonances, in a single experiment providing a complementary approach to the established proton-detected amide-based multidimensional solid-state NMR experiments for the study and resonance assignment of biosolid samples, in particular for aromatic side chain resonances.     

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.     

Band-selective 13C Homonuclear 3D Spectroscopy for Solid Proteins at High Field with Rotor-synchronized Soft Pulses

We demonstrate improved 3D ¹³C–¹³C–¹³C chemical shift correlation experiments for solid proteins, utilizing band-selective coherence transfer, scalar decoupling and homonuclear zero-quantum polarization transfer. Judicious use of selective pulses and a z-filter period suppress artifacts with a two-step phase cycle, allowing higher digital resolution in a fixed measurement time. The novel correlation of C^ali–C^ali–CX (C^ali for aliphatic carbons, CX for any carbon) reduces measurement time by an order of magnitude without sacrificing digital resolution. The experiment retains intensity from side-chain carbon resonances whose chemical shift dispersion is critical to minimize spectral degeneracy for large proteins with a predominance of secondary structure, such as β-sheet rich fibrillar proteins and α-helical membrane proteins. We demonstrate the experiment for the β1 immunoglobulin binding domain of protein G (GB1) and fibrils of the A30P mutant of α-synuclein, which is implicated in Parkinson’s disease. Selective pulses of duration comparable the rotor period give optimal performance, but must be synchronized with the spinning in non-trivial ways to minimize chemical shift anisotropy recoupling effects. Soft pulses with a small bandwidth-duration product are best for exciting the ~70 ppm bandwidth required for aliphatic-only dimensions.     

Improved pulse sequences for sequence specific assignment of aromatic proton resonances in proteins

Aromatic proton resonances of proteins are notoriously difficult to assign. Through-bond correlation experiments are preferable over experiments that rely on through-space interactions because they permit aromatic chemical shift assignments to be established independently of the structure determination process. Known experimental schemes involving a magnetization transfer across the C^β–C^γ bond in aromatic side chains either suffer from low efficiency for the relay beyond the C^δ position, use sophisticated ¹³C mixing schemes, require probe heads suitable for application of high ¹³C radio-frequency fields or rely on specialized isotopic labelling patterns. Novel methods are proposed that result in sequential assignment of all aromatic protons in uniformly ¹³C/¹⁵N labelled proteins using standard spectrometer hardware. Pulse sequences consist of routinely used building blocks and are therefore reasonably simple to implement. Ring protons may be correlated with β-carbons and, alternatively, with amide protons (and nitrogens) or carbonyls in order to take advantage of the superior dispersion of backbone resonances. It is possible to record spectra in a non-selective manner, yielding signals of all aromatic residues, or as amino-acid type selective versions to further reduce ambiguities. The new experiments are demonstrated with four different proteins with molecular weights ranging from 11 kDa to 23 kDa. Their performance is compared with that of (Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδCɛ)Hɛ pulse sequences [Yamazaki et al. (1993) J Am Chem Soc 115:11054–11055]. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Humification processes of needle litters on forest floors in Japanese cedar (Cryptomeria japonica) and Hinoki cypress (Chamaecyparis obtusa) plantations in Japan

We quantitatively clarified the early humification processes on Japanese cedar and Hinoki cypress forest floors by using a litterbag experiment and the solid-state ¹³C CPMAS NMR technique. There was no significant effect on litter mass loss during early humification between both coniferous litters regardless of the shape of their needles. Carbon composition in both litters showed similar trends during early humification. A/O-A as a humification index was low, around 0.6, in both litters throughout the experiment period although 60% of litter mass was lost. Coniferous litter incubated for 3 years might not be well-humified and would be susceptible to physical fragmentation. Carbon mass loss rates in conifers were in the following order: O-alkyl > aliphatic > aromatic > carbonyl carbons, differing with hardwoods. Conifers had concomitantly higher and lower mass loss rates of aliphatic and aromatic carbons than hardwoods. Soil organic carbon (SOC) accumulated in topsoil for conifers had relatively high and low contents of aliphatic and aromatic carbons than that for hardwood. These compositional differences of SOC among forests could be caused by the high and low supply rates of aliphatic and aromatic carbons from litter to topsoil. Consequently, initial litter nature and humification processes can affect the compositional qualities of SOC accumulated in soil.     

Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c

Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional ¹³C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Nová?ek et al. in J Biomol NMR 50(1):1–11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

Three-dimensional 1H-TOCSY-relayed ct-[13C,1H]-HMQC for aromatic spin system identification in uniformly 13C-labeled proteins

Summary Three-dimensional ¹H-TOCSY-relayed ct-[¹³C,¹H]-HMQC is a novel experiment for aromatic spin system identification in uniformly ¹³C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect ¹H and ¹³C chemical shift evolution periods and for the generation of ¹H-¹H antiphase magnetization to accelerate the ¹H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of ¹³C-¹H zero-quantum and double-quantum coherences is to first order insensitive to ¹³C-¹H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.     

Backbone 1H, 13C, and 15N NMR assignments for the Cyanothece 51142 protein cce_0567: a protein associated with nitrogen fixation in the DUF683 family

Cyanothece 51142 contains a 78-residue protein, cce_0567, that falls into the DUF683 family of proteins associated with nitrogen fixation. Here we report the assignment of most of the main chain and ¹³C^β side chain resonances of the ∼40 kDa homo-tetramer.     

3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

Editing and diagonal peak suppression in three-dimensional HCCH protein NMR correlation experiments

A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves ¹³C-¹³C COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the ¹³C-¹³C homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the ¹H-¹H (3D spectrum recorded with two ¹H and one ¹³C dimension) or the ¹³C-¹³C (3D spectrum recorded with one ¹H and two ¹³C dimensions) two-dimensional projection. The new experiment is demonstrated using a ¹³C,¹⁵N-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz.     

Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins

Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (¹H, ¹⁵N, ¹³C^α and ¹³C′) resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment—(4,3)D-hnCOCANH and exploits the linear combinations of backbone (¹³C^α and ¹³C′) chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks—which is different in sum (CA + CO) and difference (CA ? CO) frequency regions—greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide ¹⁵N and backbone ¹³C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) ¹⁵N and ¹³C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F ₂–F ₃ planes of the spectrum corresponding to sum (CA + CO) and difference (CA ? CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.      

Selective diagonal-free 13C,13C-edited aliphatic–aromatic NOESY experiment with non-uniform sampling

A band-selective aromatic–aliphatic C,C-edited four-dimensional NOESY experiment is proposed here. Its key advantage is the absence of auto-correlation signals which makes it very attractive for joint use with non-uniform sampling. It is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses (for either aromatic-13C or aliphatic-13C spins). The method was applied to the sample of E32Q mutant of human S100A1 protein, a homodimer of total molecular mass ~20 kDa. High-resolution 4D spectra were obtained from ~1.5 % of sampling points required conventionally. It is shown that superior resolution facilitates unambiguous assignment of observed aliphatic–aromatic cross-peaks. Additionally, the addition of aliphatic-13C dimension enables to resolve peaks with degenerated aliphatic ¹H chemical shifts. All observed cross-peaks were validated against previously determined 3D structure of E32Q mutant of S100A1 protein (PDB 2LHL). The increased reliability of structural constraints obtained from the proposed high-resolution 4D 13C(ali),13C(aro)-edited NOESY can be exploited in the automated protocols of structure determination of proteins.