Constant-time Evolution Period Research Articles

Sugar-to-base correlation in nucleic acids with a 5D APSY-HCNCH or two 3D APSY-HCN experiments

A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all (15)N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.

Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment

Protein backbone 15N spin relaxation rates measured by solution NMR provide useful dynamic information with a site-specific resolution. The conventional method is to record a series of 2D 1H- 15N HSQC spectra with varied relaxation delays, and derive relaxation rate from the following curve fitting on the resonance intensities. Proteins with poorly resolved spectra often require several 3D HNCO spectra to be collected on a 15N/ 13C double labeled protein sample. In order to reduce the relaxation dimension Carr et al . (P.A. Carr, D.A. Fearing, A.G. Palmer, 3D accordion spectroscopy for measuring N-15 and (CO)-Carbon-13 relaxation rates in poorly resolved NMR spectra, J. Magn. Reson. 132 (1998) 25–33) employed an Accordion type HNCO pulse sequence to obtain 15N or 13C T 1 relaxation rates by numerical fitting of the relaxation interfered free induction decay (FID) data. To avoid intensive analysis of the time domain data, we propose a modified protocol to measure 15N T 1 and T 2 relaxation rates from easily obtained line-widths in an Accordion HNCO spectrum. Both T 1 and T 2 relaxation could be simultaneously convoluted into the constant-time evolution periods of 13C′ and 15N, respectively. The relaxation delay was allowed to reach at least 3 × T 1 or 3 × T 2 so that the signal was substantially decayed by the end of the FID, and the resulting peak full-width at half height (FWHH) could be directly used to calculate relaxation rate. When applied to the 76-residue Ubiquitin and the 226-residue glutamine-binding protein (GlnBP), this method yielded T 1 and T 2 values deviating on average by 4–6% and 5–7%, respectively, from the measurements based on the conventional 2D method. In comparison, the conventional methods possessed intrinsic error ranges of 2–4% for T 1 and 3–6% for T 2. In addition to comparable accuracy, the fully-relaxed Accordion HNCO method presented here allowed measurements of relaxation rates for resonances unresolved in 2D spectra, thus providing a more complete dynamic picture of the protein.

Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groups

Rapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl “isotags” was made possible with high resolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimal correlation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupled with the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highly resolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).

Elimination of 13Cα Splitting in Protein NMR Spectra by Deconvolution with Maximum Entropy Reconstruction

Homonuclear 13C-13C couplings can significantly reduce the sensitivity and resolution of multidimensional NMR experiments. The most important of these couplings is the 13Calpha-13Cbeta coupling, and several different methods have been developed to eliminate its effect from spectra used for backbone assignment, including short or constant-time evolution periods, selectively labeled amino acids, and multiple-band decoupling sequences. In this communication we show that postacquisition deconvolution of the spectra with a maximum entropy algorithm can be superior to experimental decoupling. The method is very robust, does not introduce shifts of the resonance positions, and simplifies the measurement of the most important NMR experiments for protein backbone assignment.

Water-macromolecule interactions by NMR: a quadrature-free constant-time approach and its application to CI2.

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a square root 2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-sheet strands were identified, solving previously reported discrepancies between the X-ray and refined NMR structure of CI2. The proposed experiment in particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand-macromolecule complexes.

Phase labeling of C-H and C-C spin-system topologies: application in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments for determining backbone resonance assignments in proteins.

Triple-resonance experiments can be designed to provide useful information on spin-system topologies. In this paper we demonstrate optimized proton and carbon versions of PFG-CT-HACANH and PFG-CT-HACA(CO)NH 'straight-through' triple-resonance experiments that allow rapid and almost complete assignments of backbone H(alpha), 13C(alpha), 15N and H(N) resonances in small proteins. This work provides a practical guide to using these experiments for determining resonance assignments in proteins, and for identifying both intraresidue and sequential connections involving glycine residues. Two types of delay tunings within these pulse sequences provide phase discrimination of backbone Gly C(alpha) and H(alpha) resonances: (i) C-H phase discrimination by tuning of the refocusing period tau(a_f); (ii) C-C phase discrimination by tuning of the 13C constant-time evolution period 2T(c). For small proteins, C-C phase tuning provides better S/N ratios in PFG-CT-HACANH experiments while C-H phase tuning provides better S/N ratios in PFG-CT-HACA(CO)NH. These same principles can also be applied to triple-resonance experiments utilizing 13C-13C COSY and TOCSY transfer from peripheral side-chain atoms with detection of backbone amide protons for classification of side-chain spin-system topologies. Such data are valuable in algorithms for automated analysis of resonance assignments in proteins.

Improved NMR Experiments for the Correlation of Backbone-Carbon with Aliphatic-Proton Resonances in Proteins

We describe simple modifications of the HCACO experiment that include 13 C α and 13 CO constant time evolution periods. An overall duration of 28 ms involving carbon transverse magnetization in combination with an optimal magnetization-transfer efficiency results in a high sensitivity while providing reasonable resolution in the 13 C α domain. The improved protocol can be extended to pulse sequences which yield correlations between the 13 C α , 13 CO pair and the aliphatic side-chain proton resonances.

An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins

Recently proposed triple-resonance experiments (1-14) make it possible to obtain complete backbone assignments of isotopically enriched proteins without recourse to NOE information. A main practical problem with the triple-resonance assignment approach is the necessity to record a substantial number of 3D spectra, each requiring typically at least one or two days of measuring time. A 4D triple-resonance approach has been proposed to remove the need for recording more than just two 4D experiments for completing the backbone assignments ( 11). However, if there is overlap in the ‘H,-13C, region of the 2D spectrum, these and other (12) 4D experiments also do not offer unique connections between sequential amides. In cases of such overlap, an experiment that relays magnetization from the amide to the intraresidue carbonyl can resolve the ambiguity caused by the C,-H, degeneracy ( 13). Alternatively, an experiment named CBCA(CO)NH, which correlates amide signals with the C, and Cs reson.ances of the preceding residue by using relay via the intervening i3C0 nucleus, provides information on the type of amino acid preceding each amide and thus frequently resolves the ambiguity caused by C,-H, degeneracy (14). Here we describe a similar experiment, named CBCANH, which correlates the amide ‘H and “N signals with ‘both the intraresidue C, and C, signals and with the C, and C, of the preceding residue. For smalland medium-sized proteins, up to -2OkDa, this 3D experiment alone can yield virtually complete assignments of the backbone and the C, resonances. The pulse scheme for the CBCANH experiment is sketched in Fig. 1. Before describing the experiment in more detail, we first outline its general mechanism. An INEPT transfer enhances polarization of both C, and CB spins. Chemical shifts of both C, and CB are encoded during the constant-time evolution period, 2TAB ( 7, 8, 10, Z4). When the 90 ’ 13C pulse is applied, at time b in Fig. 1, CB magnetization is transferred to C,, but at the same time a fraction of C, magnetization remains on C,. Subsequently, C, magnetization is transferred to its intraresidue 15N and to the 15N of the next residue, via the ’ JColN and 2 JcaN couplings, respectively. Following a constant-time 15N evolution period, the “N magnetization is transferred to the amide protons with a reverse INEPT sequence, prior to detection of the HN signals.

4D NMR triple-resonance experiments for assignment of protein backbone nuclei using shared constant-time evolution periods

4D NMR triple-resonance experiments for assignment of protein backbone nuclei using shared constant-time evolution periods