Intraresidue HNCA and COHNCA experiments for protein backbone resonance assignment

Two novel experiments, intra-HNCA and intra-COHNCA, are presented for sequential backbone resonance assignment of (13)C, (15)N labeled proteins. The advantage with respect to conventional pulse schemes is the suppression of the sequential (15)N-->(13)C(alpha) coherence transfer pathway, which can be separately obtained from a HNCOCA correlation experiment. This results in a two-fold reduction of the number of detected correlation peaks. Spectral simplification is especially important for efficient automated assignment protocols as required in the context of high-throughput protein studies by NMR. The performance of the new experiments is demonstrated on an 18-kDa protein fragment of the E. coli sulfite reductase and compared to conventional techniques in terms of sensitivity and resolution.     

A set of 4D NMR experiments of enhanced resolution for easy resonance assignment in proteins

This paper presents examples of techniques based on the principle of random sampling that allows acquisition of NMR spectra featuring extraordinary resolution. This is due to increased dimensionality and maximum evolution time reached. The acquired spectra of CsPin protein and maltose binding protein were analyzed statistically with the aim to evaluate each technique. The results presented include exemplary spectral cross-sections. The spectral data provided by the proposed techniques allow easy assignment of backbone and side-chain resonances.     

iHADAMAC: a complementary tool for sequential resonance assignment of globular and highly disordered proteins

An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.     

A set of BEST triple-resonance experiments for time-optimized protein resonance assignment

A series of sequential, intra-residue, and bi-directional BEST H-N-CA, H-N-CO, and H-N-CB pulse sequences is presented that extends the BEST concept introduced recently for fast multidimensional protein NMR [Schanda et al., J. Am. Chem. Soc. 128 (2006) 9042] to the complete set of experiments required for sequential resonance assignment. We demonstrate for the protein ubiquitin that 3D BEST H-N-C correlation spectra can be recorded on a 600MHz NMR spectrometer equipped with a cryogenic probe in only a few minutes of acquisition time with sufficient sensitivity to detect all expected cross peaks.     

Optimized 3D-NMR sampling for resonance assignment of partially unfolded proteins

Resonance assignment of NMR spectra of unstructured proteins is made difficult by severe overlap due to the lack of secondary structure. Fortunately, this drawback is partially counterbalanced by the narrow line-widths due to the internal flexibility. Alternate sampling schemes can be used to achieve better resolution in less experimental time. Deterministic schemes (such as radial sampling) suffer however from the presence of systematic artifacts. Random acquisition patterns can alleviate this problem by randomizing the artifacts. We show in this communication that quantitative well-resolved spectra can be obtained, provided that the data points are properly weighted before FT. These weights can be evaluated using the concept of Voronoi cells associated with the data points. The introduced artifacts do not affect the direct surrounding of the peaks and thus do not alter the amplitude and frequency of the signals. This procedure is illustrated on 60-residue viral protein, which lacks any persistent secondary structure and thus exhibits major signal overlap.     

Novel 2D triple-resonance NMR experiments for sequential resonance assignments of proteins

We present 2D versions of the popular triple resonance HN(CO) CACB, HN(COCA)CACB, HN(CO)CAHA, and HN(COCA) CAHA experiments, commonly used for sequential resonance assignments of proteins. These experiments provide information about correlations between amino proton and nitrogen chemical shifts and the alpha- and beta-carbon and alpha-proton chemical shifts within and between amino acid residues. Using these 2D spectra, sequential resonance assignments of H(N), N, C(alpha), C(beta), and H(alpha) nuclei are easily achieved. The resolution of these spectra is identical to the well-resolved 2D (15)N-(1)H HSQC and H(NCO)CA spectra, with slightly reduced sensitivity compared to their 3D and 4D versions. These types of spectra are ideally suited for exploitation in automated assignment procedures and thereby constitute a fast and efficient means for NMR structural determination of small and medium-sized proteins in solution in structural genomics programs.     

A 'just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity

Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast 13CO transverse relaxation and passive 13Calpha-13Cbeta coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses 13Calpha-13Cbeta coupling while collecting 13CO shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective 13Calpha inversion pulses were performed using a 2H/13C/15N labeled sample of the 29 kDa HCA II protein at 15 degrees C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper 13Calpha refocusing and13Calpha-13Cbeta decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide 13CO resonance information regardless of residue type.     

Electron nuclear quadruple resonance for assignment of overlapping spectra

Multiple resonance methods are important tools in EPR for revealing the network of hyperfine levels of free radicals and paramagnetic centers. The variations of electron nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM) techniques help to correlate nuclear frequencies with each other. These methods have limited utility when there is extensive overlap or suspected overlap in the EPR spectrum between different species or different orientations. In the ENDOR spectrum, overlap and second-order shifts of lines also leads to ambiguity in assignment and interpretation. A new electron nuclear multiple resonance method is presented here that is based on population transfer ENDOR. It is a quadruple resonance method that correlates ENDOR lines and reveals the network of hyperfine levels in samples with unoriented paramagnetic species and in samples with overlapping EPR or ENDOR lines.     

Soft-triple resonance solid-state NMR experiments for assignments of U-13C, 15N labeled peptides and proteins

The process of obtaining sequential resonance assignments for heterogeneous polypeptides and large proteins by solid-state NMR (ssNMR) is impeded by extensive spectral degeneracy in these systems. Even in these challenging cases, the cross peaks are not distributed uniformly over the entire spectral width. Instead, there exist both well-resolved single resonances and distinct groups of resonances well separated from the most crowded region of the spectrum. Here, we present a series of new triple resonance experiments that exploit the non-uniform clustering of resonances in heteronuclear correlation spectra to obtain additional resolution in the more crowded regions of a spectrum. Homonuclear and heteronuclear dipolar recoupling sequences are arranged to achieve directional transfer of coherence between neighboring residues in the peptide sequence. A frequency-selective (soft) pulse is applied to select initial polarization from a limited (and potentially) well-resolved region of the spectrum. The pre-existing resolution of one or more spins is thus utilized to obtain additional resolution in the more crowded regions of the spectrum. A new protocol to utilize these experiments for sequential resonance assignments in peptides and proteins is also demonstrated.     

3D HCCH(3)-TOCSY for resonance assignment of methyl-containing side chains in (13)C-labeled proteins

Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t(2max) = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).     

3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.     

Simple and efficient photo-ionization loading of ions for precision ion-trapping experiments

We report a simple and efficient method to load a Paul trap with Ca⁺ ions. A beam of neutral atomic calcium is ionized in a two-step photo-ionization process using uv-diode lasers near 423 nm and 390 nm. Photo-ionization of a calcium beam for loading a Paul trap has first been demonstrated by Kjaergaard et al. The advantages of our method are the use of cheap and easily handled diode-laser systems and the large cross section for field ionization when exciting high-lying Rydberg states. Finally, we discuss the advantages of photo-ionization for ion generation compared to loading by electron bombardment. Received: 24 August 2001 / Revised version: 16 October 2001 / Published online: 23 November 2001     

An efficient peak assignment algorithm for two-dimensional NMR correlation spectra of framework structures

An algorithm is described for efficiently assigning the resonances in NMR spectra to the inequivalent atoms in the structure under study based on the information in two-dimensional NMR correlation experiments and the 'connectivities' known from the structure. The algorithm, which is based on basic graph theory concepts, finds all possible assignments sets which are consistent with the experimentally observed correlations and known connectivities in a very efficient manner. It is designed to deal with less than ideal experimental data in which there may be overlapping peaks and uncertainty about the presence or absence of correlation peaks. The algorithm was primarily developed for assigning the peaks in the high-resolution solid-state 29Si MAS NMR spectra of highly siliceous zeolites based on two-dimensional 29Si INADEQUATE spectra and is described using the zeolites ZSM-12 and ZSM-5 as working examples. Peak assignment for zeolite frameworks is particularly challenging since there is often little or no information to distinguish peaks from one another such as characteristic chemical shifts, relative intensities, or different relaxation times. The algorithm may be a useful tool for easily, reliably, and efficiently working out peak assignments from other types of correlation experiments on other types of systems and further examples are provided in the Supplementary material.     

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

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

The rate memory of a polymer close toT gas elucidated by reduced 4-D NMR echo experiments

We analyze the reorientational dynamics of a polymer close to the glass transition. Via comparison of two-time and four-time correlation functions, obtained from multidimensional nuclear magnetic resonance¹³C echo experiments, we are particularly sensitive to the fluctuations within the heterogeneous distribution of mobilities. From variation of the experimental parameters we obtain a detailed picture of the interplay between jump and diffusion dynamics, including the different types of motional heterogeneities and the geometry of the system.     

Tailored HCCH-TOCSY experiment for resonance assignment in the proximity of a paramagnetic center

The presence of a paramagnetic center may disturb both coherent and incoherent communication between nuclear spins that are affected, to some extent, by the hyperfine interaction. This is a limiting factor to an extensive use of paramagnetic probes in NMR spectroscopy to enhance partial alignment and to exploit cross correlation effects and pseudocontact shifts. We propose here an HCCH-TOCSY experiment tailored to identify spin systems involving resonances that are partly or completely affected by hyperfine interaction. The efficiency of polarization transfer steps when fast relaxing nuclei are involved is discussed. The sequence is tested for the protein Calbindin D(9k), in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion as well as for the oxidized form of cytochrome b(562).     

H-start for exclusively heteronuclear NMR spectroscopy: The case of intrinsically disordered proteins

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on ¹³C direct detection, which exploit the ¹H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.     

High-throughput backbone resonance assignment of small 13C,15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J(1H,13C): HNCACBcodedHAHB

The proposed three-dimensional triple-resonance experiment HNCACBcodedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13Calpha, 13Cbeta, 1Halpha, and 1Hbeta. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1Halpha and 1Hbeta chemical shifts are then coded in the line shape of the cross-peaks of 13Calpha, 13Cbeta along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACBcodedHAHB experiment was applied to approximately 85% labeled 13C,15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.     

Five-level k · p model for conduction electrons in GaAs. Description of cyclotron resonance experiments

Five-level k·p model for the conduction electrons in GaAs in the presence of a quantising magnetic field is developed and used to describe spin splittings of the cyclotron resonance and the donor-shifted cyclotron resonance peaks, observed in this material up to fields of 22.5 T. It is shown that the spin splittings are insensitive to polaron effects and that their values can be very well described by the model.Required band parameters correctly account for the rate of electron spin relaxation in GaAs due to inversion asymmetry, as determined by other authors.