Homonuclear 13C Research Articles

High-Speed Tracer Analysis of Metabolism (HS-TrAM).

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) offers high resolution data, yet is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting due to J-coupling in 1H, 13C-HSQC NMR spectra, which allow the rapid collection of NMR data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these novel techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ, and also show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy.

In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

AbstractIn this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

Spectral editing for in vivo 13C magnetic resonance spectroscopy

In vivo detection of carboxylic/amide carbons is a promising technique for studying cerebral metabolism and neurotransmission due to the very low RF power required for proton decoupling. In the carboxylic/amide region, however, there is severe spectral overlap between acetate C1 and glutamate C5, complicating studies that use acetate as an astroglia-specific substrate. There are no known in vivo MRS techniques that can spectrally resolve acetate C1 and glutamate C5 singlets. In this study, we propose to spectrally separate acetate C1 and glutamate C5 by a two-step J-editing technique after introducing homonuclear 13C– 13C scalar coupling between carboxylic/amide carbons and aliphatic carbons. By infusing [1,2- 13C 2]acetate instead of [1- 13C]acetate the acetate doublet can be spectrally edited because of the large separation between acetate C2 and glutamate C4 in the aliphatic region. This technique can be applied to studying acetate transport and metabolism in brain in the carboxylic/amide region without spectral interference.

Sensitizing solid state nuclear magnetic resonance of dilute nuclei by spin-diffusion assisted polarization transfers

Recent years have witnessed efforts geared at increasing the sensitivity of NMR experiments, by relying on the suitable tailoring and exploitation of relaxation phenomena. These efforts have included the use of paramagnetic agents, enhanced (1)H-(1)H incoherent and coherent transfers processes in 2D liquid state spectroscopy, and homonuclear (13)C-(13)C spin diffusion effects in labeled solids. The present study examines some of the opportunities that could open when exploiting spontaneous (1)H-(1)H spin-diffusion processes, to enhance relaxation and to improve the sensitivity of dilute nuclei in solid state NMR measurements. It is shown that polarization transfer experiments executed under sufficiently fast magic-angle-spinning conditions, enable a selective polarization of the dilute low-γ spins by their immediate neighboring protons. Repolarization of the latter can then occur during the time involved in monitoring the signal emitted by the low-γ nuclei. The basic features involved in the resulting approach, and its potential to improve the effective sensitivity of solid state NMR measurements on dilute nuclei, are analyzed. Experimental tests witness the advantages that could reside from utilizing this kind of approach over conventional cross-polarization processes. These measurements also highlight a number of limitations that will have to be overcome for transforming selective polarization transfers of this kind into analytical methods of choice.

Broadband 13C-Homodecoupled Heteronuclear Single-Quantum Correlation Nuclear Magnetic Resonance

13C-homodecoupling in F1: Eliminating homonuclear 13C scalar couplings is a challenging NMR problem when enriched compounds are studied by NMR. The 13C-homodecoupled heteronuclear single-quantum correlation (HSQC) experiment (BBHD-HSQC) relies on the Zangger–Sterk pulse sequence element to combine spatial encoding of the chemical shift with the refocusing of couplings. The sequence is applied to fully enriched 13C cholesterol.

Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear 15N– 13C α and homonuclear 13C– 13C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped 13C α resonances by strategically labeling the preceding residue. This method is highly complementary to 13 C i - 1 ′ - 15 N i - 13 C i α and 13 C i - 1 α - 15 N i - 1 - 13 C i ′ experiments to distinguish inter-residue spin systems at a minimal cost to signal-to-noise.

Dipolar-coupling-mediated total correlation spectroscopy in solid-state 13C NMR: Selection of individual 13C– 13C dipolar interactions

Herein is described a useful approach in solid-state NMR, for selecting homonuclear 13C– 13C spin pairs in a multiple- 13C homonuclear dipolar coupled spin system. This method builds upon the zero-quantum (ZQ) dipolar recoupling method introduced by Levitt and coworkers (Marin-Montesinos et al., 2006 [30]) by extending the originally introduced one-dimensional (1D) experiment into a two-dimensional (2D) method with selective irradiation scheme, while moving the 13C– 13C mixing scheme from the transverse to the longitudinal mode, together with a dramatic improvement in the proton decoupling efficiency. Selective spin-pair recoupling experiments incorporating Gaussian and cosine-modulated Gaussian pulses for inverting specific spins were performed, demonstrating the ability to detect informative, simplified/individualized, long-range 13C– 13C homonuclear dipolar coupling interactions more accurately by removing less informative, stronger, short-range 13C– 13C interactions from 2D correlation spectra. The capability of this new approach was demonstrated experimentally on uniformly 13C–labeled Glutamine and a tripeptide sample, GAL.

Labeling strategies for 13C-detected aligned-sample solid-state NMR of proteins

13C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13C sites so that dipole–dipole couplings among the 13C are minimized, thus eliminating the need for homonuclear 13C– 13C decoupling in either indirect or direct dimensions of one- or multi-dimensional NMR experiments that employ 13C detection. The optimal percentage for random fractional 13C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on [2- 13C]-glucose-containing media was found to be most effective.

Dipolar Chemical Shift Correlation Spectroscopy for Homonuclear Carbon Distance Measurements in Proteins in the Solid State: Application to Structure Determination and Refinement

High-resolution solid-state NMR spectroscopy has become a promising tool for protein structure determination. Here, we describe a new dipolar-chemical shift correlation experiment for the measurement of homonuclear 13C-13C distances in uniformly 13C,15N-labeled proteins and demonstrate its suitability for protein structure determination and refinement. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). Both intraresidue and interresidue distances between carbonyl atoms and atoms in the aliphatic side chains were collected using a three-dimensional chemical shift correlation spectroscopy experiment that uses homogeneously broadened rotational resonance recoupling for carbon mixing. A steady-state approximation for the polarization transfer function was employed in data analysis, and a total of 100 intramolecular distances were determined, all in the range 2.5-5.5 A. An additional 41 dipolar contacts were detected, but the corresponding distances could not be accurately quantified. Additional distance and torsional restraints were derived from the proton-driven spin diffusion measurements and from the chemical shift analysis, respectively. Using all these restraints, it was possible to refine the structure of GB1 to a root-mean square deviation of 0.8 A. The approach is of general applicability for peptides and small proteins and can be easily incorporated into a structure determination and refinement protocol.

Triple resonance experiments for aligned sample solid-state NMR of 13C and 15N labeled proteins

Initial steps in the development of a suite of triple-resonance 1H/ 13C/ 15N solid-state NMR experiments applicable to aligned samples of 13C and 15N labeled proteins are described. The experiments take advantage of the opportunities for 13C detection without the need for homonuclear 13C/ 13C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ∼20% randomly labeled with 13C in all backbone and side chain carbon sites and ∼100% uniformly 15N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13C labeled at only the α-carbon and 15N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13C/ 13C decoupling while detecting 13C signals is avoided in the first case because of the low probability of any two 13C nuclei being bonded to each other; in the second case, the labeled 13C α sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13C chemical shift and 1H– 13C and 15N– 13C heteronuclear dipolar coupling frequencies associated with the 13C α and 13C′ backbone sites, which provide orientation constraints complementary to those derived from the 15N labeled amide backbone sites. 13C/ 13C spin-exchange experiments identify proximate carbon sites. The ability to measure 13C– 15N dipolar coupling frequencies and correlate 13C and 15N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13C chemical shift and 1H– 13C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.

Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteins.

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different (13)C labeling schemes with the purpose of overcoming the interfering effects of (13)C-(13)C dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% (13)C in all sites and metabolically labeled from bacterial growth on media containing [2-(13)C]-glycerol or [1,3-(13)C]-glycerol enables direct (13)C detection in solid-state NMR experiments without the need for homonuclear (13)C-(13)C dipole-dipole decoupling. The (13)C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent (15)N-detected spectra. The isotopic labeling pattern was analyzed for [2-(13)C]-glycerol and [1,3-(13)C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored (13)C-labeled samples, in contrast to their failure with conventional 100% uniformly (13)C-labeled samples.

Asymmetry in 13C−13C COSY Spectra Provides Information on Ligand Geometry in Paramagnetic Proteins

The relative intensity of Calpha-C' cross-peaks in homonuclear 13C COSY spectra depends on the relaxation properties of Calpha and C' spins, which, in the proximity of a paramagnetic center, are related to the metal-to-carbon distance. Their quantitative analysis has lead, for the cerium-substituted dicalcium protein, calbindin D9k, to the straightforward identification of peaks arising from metal-coordinating groups. The monodentate or bidentate metal binding mode of carboxylates was identified directly via NMR.

Homo-nuclear 13C J-decoupling in uniformly 13C-enriched solid proteins

Recently, we reported an analysis of carbon lineshapes in high resolution solid-state NMR spectra of uniformly 13C-enriched amino acids. Application of a 13C J-decoupling protocol during the carbon chemical shift evolution period allowed us to separate the contribution of the second-order dipolar shift from that of the 13C– 13C J-coupling interactions to carbon linewidths. In this work, we have extended this approach to microcrystalline proteins. We describe the performance of the J-decoupling sequence applied to remove homo-nuclear 13C J-couplings in the 13C spectra of ubiquitin. Analysis of the J-decoupling efficiency for Cα and carbonyl protein sites showed that a significant gain in resolution can be achieved.

Magic-Angle Spinning Nuclear Magnetic Resonance under Ultrahigh Field Reveals Two Forms of Intermolecular Interaction within CH2Cl2-Treated (3R)-Type BacteriochlorophyllcSolid Aggregate

(31R)-bacteriochlorophyll (BChl) c solid aggregates with an absorbance around 740 nm were formed from BChl c dimers, and 2-D homonuclear 13C−13C radio frequency-driven dipolar recoupling as well as proton-driven spin diffusion dipolar correlation NMR spectra have been collected in ultrahigh magnetic field. Doubling of signals is observed for most carbons in the BChl c macrocycle, leading to two correlation networks. In this way, two major fractions denoted types A and B are identified. Some of the ring carbons show multiple resonances, revealing additional slight differences in microstructural environment. 2-D heteronuclear 1H−13C correlation data have been recorded using the frequency- and phase-switched Lee−Goldburg technique to assign the 1H response. 15N chemical shifts are assigned from 2-D heteronuclear 15N−13C correlation experiments using spectrally induced filtering in combination with cross polarization. Also the nitrogen atoms in the pyrrole rings I, II, and IV (NI, NII, and NIV, respectively) ...

Rotational-echo double resonance of uniformly labeled 13C clusters

The use of rotational-echo double resonance NMR to measure distances from an observed tightly coupled cluster of 13C spins to a distant 15N, 31P, or 19F is practical if 13C chemical shifts and homonuclear 13C– 13C isotropic J interactions are refocused by a combination of rotor-synchronized 13C π and π/2 pulses. This scheme is illustrated by experiments performed on diluted and recrystallized l-[ 13C 3, 15N]alanine and l-[ 13C 6,α- 15N]histidine.

Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs.

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear 13C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any 13C-edited multidimensional NMR experiment. As an example we have chosen the 13C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.

2D and 3D15N−13C−13C NMR Chemical Shift Correlation Spectroscopy of Solids: Assignment of MAS Spectra of Peptides

Strategies are discussed for resolving and assigning peptide backbone and side chain resonances in uniformly 13C,15N-labeled solid peptides. Methods for 2D 13C−13C, 15N−(13C)−13C, and 3D 15N−13C−13C chemical shift correlation spectroscopy are demonstrated in the chemotactic tripeptide N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (MLF). Band-selective heteronuclear double-cross polarization (DCP) and γ-encoded homonuclear double-quantum mixing provide large improvements in sensitivity relative to previously published methods. Directional transfers from amide 15N to 13C‘ or 13Cα resonances provide two- to 3-fold improvements in signal intensity on the observed 13C spin, in comparison to broadband DCP. Similarly, homonuclear 13C−13C transfer is enhanced by use of the rotating frame sequence SPC-5; backbone-to-side chain polarization transfers are achieved with especially high efficiency. Furthermore, the double-quantum nature of the homonuclear transfer permits straightforward classification of C‘, Cα, Cβ, and Cγ signals, on the basis of the sign of the cross-peaks. The experiments described here include optimized solid-state analogues of the solution-state schemes commonly employed for amino acid identification and sequential backbone assignment. We expect that these experiments will facilitate application of 2D and 3D correlation methods to assignment of larger solid peptides and proteins.

Application of Site Specific 13C Enrichment and 13C NMR Spectroscopy for the Elucidation of the Formation Pathway Leading to a Red 1H-Pyrrol-3(2H)-one during the Maillard Reaction of Furan-2-carboxaldehyde and l-Alanine

The intensely red compounds (S)-4[(E)-1-formyl-2-(2-furyl)ethenyl)]-5-(2-furyl)-2-[(E)-(2-furyl)methylidene]-2,3-dihydro-α-methyl-3-oxo-1H-pyrrole-1-acetic acid (1a) and its 2-[(Z)-(2-furyl)methylene] isomer (2b) have recently been identified as the main colored compounds formed upon thermal treatment of an aqueous solution of furan-2-carboxaldehyde and l-alanine. For clarification of their formation pathways, a labeling experiment with synthetic site specific 13C-enriched furan-2-carboxaldehyde was performed. The labeled carbon positions in 1a were unequivocally assigned by 1H broad band decoupled 13C NMR spectroscopy. In contrast to conventional labeling experiments, prior to the reaction with l-alanine, the 13C-labeled furan-2-carboxaldehyde was diluted with the natural 13C abundant analogue to suppress the spectrum complexity due to homonuclear 13C−13C spin couplings. This powerful technique has been successfully used for the first time to clarify the formation route of a colored Maillard reaction pro...

Determination of Homonuclear 13C−13C J Couplings between Aliphatic Carbon Atoms in Perdeuterated Proteins

Determination of Homonuclear ¹³C−¹³C <i>J</i> Couplings between Aliphatic Carbon Atoms in Perdeuterated Proteins