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.     

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.     

The DQ-HN[CACB] and DQ-HN(CO)[CACB] sequences with evolution of double quantum Calpha-Cbeta coherences

The new variant of known HNCACB and HN(CO)CACB techniques is proposed that employs excitation and evolution of double quantum Calpha-Cbeta coherences. The most important features of the new method are: increased signal dispersion, lack of splittings due to 1J(Calpha-Cbeta) spin-spin couplings, and absence of accidental cancellations of positive and negative signals. The acquisition of both DQ-HN[CACB] and DQ-HN(CO)[CACB] techniques enables sequential assignment of protein backbone, using only Calpha-Cbeta DQ-frequencies. The determination of all Calpha and Cbeta chemical shifts requires, however, a comparison with HN(CO)CA or HNCA spectra. Examples of applications of the DQ-HN[CACB] and DQ-HN(CO)[CACB] experiments are presented, employing the 2D Reduced Dimensionality approach for 13C, 15N-labeled ubiquitin, and the 3D acquisition for 13C, 15N-double labeled Ca2+ -binding bovine S100A1 protein in the apo state (21 kDa) with overall correlation time of 8.1 ns.     

A high sensitivity 3D experiment for measuring Calpha-Halpha residual dipolar coupling constants

A new sensitivity improved approach is presented to measure the Calpha-Halpha scalar and dipolar coupling constants in 13C/15N-labeled proteins using a HA(CA)CONH scheme. The proposed experiment has significantly higher sensitivity than the previously published (HA)CA(CO)NH sequence, and provides accurate and straightforward measurements of the scalar and residual dipolar coupling constants. The sequence is easy to implement, and has been demonstrated on the C-terminal domain of the human Ku-80 protein (152 amino acid residues). On average, sensitivity is improved by 40% for both isotropic and anisotropic samples. The sensitivity enhancement is more pronounced for structured regions than unstructured regions, with an average of 50-60% enhancement being observed in the well-structured regions of the protein.     

The Influence of the Radiofrequency Excitation and Conversion Pulses on the Lineshapes and Intensities of the Triple-Quantum MAS NMR Spectra of I = 3/2 Nuclei

A rigorous examination of the various multiple-quantum magic angle spinning sequences is carried out with reference to sensitivity enhancement in the isotropic dimension and the lineshapes of the corresponding MAS peaks in the anisotropic dimension. An echo efficiency parameter is defined here, which is shown to be an indicator of the performance aspects of the various sequences. This can be used in the design of further new experiments in this field. A consequence of such a systematic analysis has been the combination of a spin-lock pulse for excitation of multiple-quantum coherences and an amplitude-modulated pulse for their conversion to observable single-quantum coherences. This approach has resulted in an improved performance over other sequences with respect to both the anisotropic lineshapes and the isotropic intensities.     

The influence of the radiofrequency excitation and conversion pulses on the lineshapes and intensities of the triple-quantum MAS NMR spectra of I = 3/2 nuclei

A rigorous examination of the various multiple-quantum magic angle spinning sequences is carried out with reference to sensitivity enhancement in the isotropic dimension and the lineshapes of the corresponding MAS peaks in the anisotropic dimension. An echo efficiency parameter is defined here, which is shown to be an indicator of the performance aspects of the various sequences. This can be used in the design of further new experiments in this field. A consequence of such a systematic analysis has been the combination of a spin-lock pulse for excitation of multiple-quantum coherences and an amplitude-modulated pulse for their conversion to observable single-quantum coherences. This approach has resulted in an improved performance over other sequences with respect to both the anisotropic lineshapes and the isotropic intensities.     

Optimization of three-dimensional TROSY-type HCCH NMR correlation of aromatic (1)H-(13)C groups in proteins.

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394-6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the (13)C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S(3) TPPI). The new 3D S(3) TPPI TROSY HCCH method is demonstrated on a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.     

Magnetic trapping and Zeeman relaxation of NH (X3Sigma-)

NH radicals are magnetically trapped and their Zeeman relaxation and energy transport collision cross sections with helium are measured. Continuous buffer-gas loading of the trap is direct from a room-temperature molecular beam. The Zeeman relaxation (inelastic) cross section of magnetically trapped electronic, vibrational, and rotational ground state NH molecules in collisions with 3He is measured to be 3.8+/-1.1 x 10(-19) cm(2) at 710 mK. The NH-He energy transport cross section is also measured, indicating a ratio of diffusive to inelastic cross sections of gamma=7 x 10(4), in agreement with recent theory [R. V. Krems, H. R. Sadeghpour, A. Dalgarno, D. Zgid, J. K?os, and G. Cha?asiński, Phys. Rev. A 68, 051401 (2003)10.1103/PhysRevA.68.051401].     

A new multi-quantum version of the HBHA(CBCACO)NH experiment with enhanced sensitivity for partially deuterated samples

A new multi-quantum version of the HBHA(CBCACO)NH experiment for partially deuterated protein samples is presented. The method is based on the significant reduction of the proton and carbon relaxation rates due to multi-quantum delays in highly deuterated proteins recently published by our group. The introduction of a multi-quantum period in the coherence transfer pathway of the HBHA(CBCACO)NH experiment yields a dramatic increase of sensitivity-on average 46% with a 75% deuterated sample of the homodimeric 31 kDa E. coli IIAMan domain. Additional resolution in the proton dimension can be achieved by a double time shared approach keeping the 1H single-quantum period at a minimum.     

季铵盐型双子表面活性剂16-4-16聚集状态的NMR研究

核磁共振弛豫，自扩散以及2D NOESY谱研究结果表明：双子表面活性剂16-4-16溶液在形成胶束的过程中，联结基团及其邻近的碳氢链质子形成胶束的壳层，而距离离子头较远的疏水质子位于胶束的内部. 与对应的单链的表面活性剂CTAB相比，其分子运动更受限制. 2D NOESY谱显示联接基团及临近的碳氢链的质子间有较强的交叉峰，表明形成胶束时，分子在联结基团附近堆积的较为紧密. 由2D NOESY谱计算得到的质子间距与HYPERCHEM模拟值有偏差，表明这些强交叉峰是分子间相互作用的结果，并且对应质子对在双子表面活性剂16-4-16分子中位于邻近的区域. 因此我们推测，双子表面活性剂16-4-16分子在球形胶束中形成特殊的排列方式.     

Very simple combination of TROSY, CRINEPT and multiple quantum coherence for signal enhancement in an HN(CO)CA experiment for large proteins

Sensitivity enhancement in liquid state nuclear magnetic resonance (NMR) triple resonance experiments for the sequential assignment of proteins is important for the investigation of large proteins or protein complexes. We present here the 3D TROSY-MQ/CRINEPT-HN(CO)CA which makes use of a 1?N-1H-TROSY element and a 13C'-13CA CRINEPT step combined with a multiple quantum coherence during the 13CA evolution period. Because of the introduction of these relaxation-optimized elements and 10 less pulses required, when compared with the conventional TROSY-HN(CO)CA experiment an average signal enhancement of a factor of 1.8 was observed for the membrane protein-detergent complex KcsA with a rotational correlation time τ(c) of around 60 ns.     

3D HSQC-HSQMBC--increasing the resolution of long-range proton-carbon correlation experiments

A 3D HSQC-HSQMBC experiment is proposed for increasing the separation of proton-carbon long-range correlation cross peaks, the lack of which is occasionally seen in corresponding 2D experiments. It is aimed at complex molecules with many protonated carbons exhibiting a narrow spread of 13C chemical shifts e.g., complex carbohydrates. It does not yield long-range correlation of quaternary carbons. An extra indirectly detected 1H dimension of this experiment provides additional separation of long-range correlation cross peaks by utilising the chemical shifts of protons directly attached to 13C. Evolution of single-quantum coherences throughout the entire pulse sequence ensures that the cross peaks are inphase pure absorption singlets in both indirectly detected dimensions, thus maximising the resolution and sensitivity of the experiment. Partial signal cancellation can be expected due to the antiphase character of peaks in the directly detected dimension. The intensity of cross peaks depends on the length of a single long-range evolution interval and values of both active and passive long-range coupling constants of each carbon. The 3D HSQC-HSQMBC experiment provided high quality long-range correlation spectra of a 2 mg pentasaccharide sample in 27 h. The technique can also be used for measurement of long-range heteronuclear coupling constants from pure antiphase multiplets in the directly detected dimension.     

Rapid assignment of protein side chain resonances using projection-reconstruction of (4,3)D HC(CCO)NH and intra-HC(C)NH experiments

The reconstruction of higher-dimensional NMR spectra from projections can provide significant savings in instrument time. Here, we demonstrate its application to the (4,3)D HC(CCO)NH and intra-HC(C)NH experiments. The latter experiment contains a novel intra-residue filter element, which selectively correlates the side chain resonances with the corresponding intra-residue amide resonances. Compared to the conventional HC(C)NH experiment, the intra-HC(C)NH experiment reduces the spectral complexity and thus the minimum number of projections required for artifact-free reconstruction by half. The use of the projection-reconstruction technique allows rapid data collection and unambiguous assignment of aliphatic side chain nuclei at high resolution.     

3D HSQC-HSQMBC—increasing the resolution of long-range proton–carbon correlation experiments

A 3D HSQC-HSQMBC experiment is proposed for increasing the separation of proton–carbon long-range correlation cross peaks, the lack of which is occasionally seen in corresponding 2D experiments. It is aimed at complex molecules with many protonated carbons exhibiting a narrow spread of ¹³C chemical shifts e.g., complex carbohydrates. It does not yield long-range correlation of quaternary carbons. An extra indirectly detected ¹H dimension of this experiment provides additional separation of long-range correlation cross peaks by utilising the chemical shifts of protons directly attached to ¹³C. Evolution of single-quantum coherences throughout the entire pulse sequence ensures that the cross peaks are inphase pure absorption singlets in both indirectly detected dimensions, thus maximising the resolution and sensitivity of the experiment. Partial signal cancellation can be expected due to the antiphase character of peaks in the directly detected dimension. The intensity of cross peaks depends on the length of a single long-range evolution interval and values of both active and passive long-range coupling constants of each carbon. The 3D HSQC-HSQMBC experiment provided high quality long-range correlation spectra of a 2 mg pentasaccharide sample in 27 h. The technique can also be used for measurement of long-range heteronuclear coupling constants from pure antiphase multiplets in the directly detected dimension.     

Deuteron spin-lattice relaxation in partly and fully deuterated (NH(4))(2)PdCl(6)

Deuteron spin-lattice relaxation and spectra were studied in partially and fully deuterated (NH(4))(2)PdCl(6) in the temperature range 5-300K. The relaxation rate maximum was observed at 45K in (ND(4))(2)PdCl(6). Its value is reduced due to limited jumps by about 33% relative to the theoretical value expected for threefold reorientations. Limited jumps correspond to an N-D vector jumping between six directions on a cone around a Pd-N vector, the angle between the N-D and Pd-N vectors being denoted Delta. This motion makes a part of the quadrupole interaction ineffective in relaxation thus reducing the maximum rate at 45K. The observed reduction leads to the value Delta=21( composite function). Limited jumps are quenched to a large extent at the order-disorder phase transition and consequently a decrease is observed in the rate. Below the transition ND(4)(+) ions reorient between the tetrahedral orientations of the ordered phase, therefore the quadrupole interaction has the full relaxing efficiency. In the 10% deuterated sample the temperature of the rate maximum is shifted to 35K and below 20K the rate itself is one order of magnitude larger than in (ND(4))(2)PdCl(6). The increase is related to (1) the absence of the order-disorder phase transition and (2) to the enhanced mobility of NH(3)D(+) because of its electric dipole moment. Limited jumps are claimed to be the dominant relaxation mechanism below 20K. The relaxation in the disordered 30% deuterated sample is quite similar to that in 10% sample. The 50% and 70% deuterated samples undergo a transition to the ordered phase. The relaxation is biexponential with the characteristic rates somewhat smaller than those in (ND(4))(2)PdCl(6), but approaching them with increasing deuteration. This variation can be explained with different mobilities and varying relative numbers of the various isotopomers NH(4-n)D(n)(+), n=1-4.     

The CLIP/CLAP-HSQC: pure absorptive spectra for the measurement of one-bond couplings

Heteronuclear residual dipolar one-bond couplings of organic molecules at natural abundance are most easily measured using t2 coupled HSQC spectra. However, inevitably mismatched transfer delays result in phase distortions due to residual dispersive antiphase coherences in such experiments. In this article, slightly modified t2 coupled HSQC experiments with clean inphase (CLIP) multiplets are introduced which also reduce the intensities of undesired long-range cross peaks. With the corresponding antiphase (CLAP) experiment, situations where alpha and beta components overlap can be resolved for all multiplicities in an IPAP manner. A comparison of the experiments using hard pulses and shaped broadband excitation and inversion pulses on the heteronucleus is given and potential spectral artefacts are discussed in detail.     

Sensitivity enhancement of HCACO by using an HMQC magnetization transfer scheme

Previous theoretical calculations have demonstrated that the multiquantum relaxation rate of (1)H(alpha)-(13)C(alpha)(R(MQ)) is, on average, 1.3 +/- 0.4 or 1.7 +/- 0.6 times slower than the single-quantum relaxation rate of (13)C(alpha)(R(C)) for a sample with or without, respectively, amide protons. By taking advantage of this fact and by using the PEP sensitivity enhancement scheme, an HMQC version of the HCACO experiment has been developed. We demonstrate that this new experiment is 23 and 55% more sensitive than the original HSQC version of the HCACO experiment, at constant times of 7 and 27 ms, respectively, for a sample of the BC domain of the ciliary neurotrophic factor receptor protein dissolved in D(2)O at 20 degrees C.     

Consequences of (129)Xe-(1)H cross relaxation in aqueous solutions.

We have investigated the transfer of polarization from (129)Xe to solute protons in aqueous solutions to determine the feasibility of using hyperpolarized xenon to enhance (1)H sensitivity in aqueous systems at or near room temperatures. Several solutes, each of different molecular weight, were dissolved in deuterium oxide and although large xenon polarizations were created, no significant proton signal enhancement was detected in l-tyrosine, alpha-cyclodextrin, beta-cyclodextrin, apomyoglobin, or myoglobin. Solute-induced enhancement of the (129)Xe spin-lattice relaxation rate was observed and depended on the size and structure of the solute molecule. The significant increase of the apparent spin-lattice relaxation rate of the solution phase (129)Xe by alpha-cyclodextrin and apomyoglobin indicates efficient cross relaxation. The slow relaxation of xenon in beta-cyclodextrin and l-tyrosine indicates weak coupling and inefficient cross relaxation. Despite the apparent cross-relaxation effects, all attempts to detect the proton enhancement directly were unsuccessful. Spin-lattice relaxation rates were also measured for Boltzmann (129)Xe in myoglobin. The cross-relaxation rates were determined from changes in (129)Xe relaxation rates in the alpha-cyclodextrin and myoglobin solutions. These cross-relaxation rates were then used to model (1)H signal gains for a range of (129)Xe to (1)H spin population ratios. These models suggest that in spite of very large (129)Xe polarizations, the (1)H gains will be less than 10% and often substantially smaller. In particular, dramatic (1)H signal enhancements in lung tissue signals are unlikely.     

Infrared Spectrum of Pyromellitic Diimide HN(CO)2C6H2(CO)2NH

The IR spectrum of pyromellitic diimide, HN(CO)₂C₆H₂(CO)₂NH, has been measured for polycrystalline sample. The observed bands have been assigned to the IR active fundamentals and combinations on the basis of a D_2h molecular symmetry by comparison with those of pyromellitic dianhydride. Band shifts from dianhydride to diimide were observed for the 0[dbnd]C-X-C[dbnd]O fundamentals and the N-H modes have been assigned by comparison with those reported for phthalimides.     

Selective dephasing of OH and NH proton magnetization based on (1)H chemical-shift anisotropy recoupling

A method for selectively suppressing the signals of OH and NH protons in (1)H combined rotation and multiple-pulse spectroscopy (CRAMPS) and in (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra is presented. It permits distinction of overlapping CH and OH/NH proton signals, based on the selective dephasing of the magnetization of OH and NH protons by their relatively large (1)H chemical-shift anisotropies. For NH protons, the (14)N-(1)H dipolar coupling also contributes significantly to this dephasing. The dephasing is achieved by a new combination of heteronuclear recoupling of these anisotropies with (1)H homonuclear dipolar decoupling. Since the 180 degrees pulses traditionally used for heteronuclear dipolar and chemical-shift anisotropy recoupling would result in undesirable homonuclear dephasing of proton magnetization, instead the necessary inversion of the chemical-shift Hamiltonian every half rotation period is achieved by inverting the phases of all the pulses in the HW8 multiple-pulse sequence. In the HETCOR experiments, carefully timed (13)C 180 degrees pulses remove the strong dipolar coupling to the nearby (13)C spin. The suppression of NH and OH peaks is demonstrated on crystalline model compounds. The technique in combination with HETCOR NMR is applied to identify the CONH and NH-CH groups in chitin and to distinguish NH and aromatic proton peaks in a peat humin.