Measurement of long-range 1H-1H dipolar couplings in weakly aligned proteins

Measurement of 1H-1H dipolar couplings in macromolecules, weakly oriented by a dilute liquid crystalline medium, is generally limited to the largest such interactions. By removing dipolar couplings to nearest neighbors, either by decoupling, deuteration, or both, more remote interactions become accessible. The approach is demonstrated for measurement of amide-amide interactions in the proteins calmodulin and ubiquitin and permits observation of direct dipolar couplings between protons up to 7 A apart. Quantitative evaluation of 1H-1H dipolar couplings measured in ubiquitin shows excellent agreement with its solution structure.     

Pseudomultidimensional NMR by spin-state selective off-resonance decoupling

An alternate technique for accurately monitoring the chemical shift in multidimensional NMR experiments using spin-state selective off-resonance decoupling is presented here. By applying off-resonance decoupling on spin S during acquisition of spin I, we scaled the scalar coupling J(I,S) between the spins, and the residual scalar coupling turns out to be a function of the chemical shift of spin S. Thus, the chemical shift information of spin S is indirectly retained, without an additional evolution period and the accompanying polarization transfer elements. The detection of the components of the doublet using spin-state selection enables an accurate measurement of the residual scalar coupling and a precise value for the chemical shift, concomitantly. The spin-state selection further yields two subspectra comprising either one of the two components of the doublet and thereby avoiding the overlap problems that arise from off-resonance decoupling. In general, spin-state selective off-resonance decoupling can be incorporated into any pulse sequence. Here, the concept of spin-state selective off-resonance decoupling is applied to 3D (13)C or (15)N-resolved [(1)H,(1)H]-NOESY experiments, adding the chemical shift of the heavy atom attached to the hydrogen ((13)C or (15)N nuclei) with high resolution resulting in a pseudo-4D. These pseudo-4D heavy-atom resolved [(1)H, (1)H]-NOESY experiments contain chemical shift information comparable to that of 4D (13)C or (15)N-resolved [(1)H,(1)H]-NOESY, but with an increase in chemical shift resolution by 1-2 orders of magnitude.     

HOBS methods for enhancing resolution and sensitivity in small DNA oligonucleotide NMR studies

¹H NMR spectra from biopolymers give chemical shifts classified according to proton type and often suffer from signal degeneracy. Data from nucleic acids are particularly prone to this failing. Recent developments in proton broadband decoupling techniques with the promise of enhanced resolution at full sensitivity have allowed us to investigate the application of homonuclear band‐selective (HOBS) decoupling to the study of small synthetic DNA molecules and to compare these with results from classical and pure shift techniques. Improved signal resolution at full sensitivity in both HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data is reported for three example small DNA molecules. Comparisons of ¹H T₁ and integrals of signals from HOBS‐1D ¹H and HOBS‐2D [¹H, ¹H] NOESY NMR data with those of standard data collection methods are also reported. The results show that homonuclear HOBS‐NOESY data are useful for data assignment purposes and have some merit for quantification purposes. In general, we show that resolution and sensitivity enhancement of ¹H NMR data for small DNA samples may be achieved without recourse to higher magnetic field strength at full sensitivity in a band‐selected manner. Copyright © 2014 John Wiley & Sons, Ltd.     

High resolution 13C spectroscopy by Fourier transform NMR. Thiete sulfone

The high resolution ¹³C NMR spectrum of thiete sulfone (obtained without wide-band ¹H decoupling) indicates that ring strain and re-hybridization in this small ring compound are significant. Pulsed-Fourier operation allows rapid determination of spectral parameters. Experimental acquisition time by pulsed NMR without ¹H decoupling is comparable to the experimental time required for swept ¹³C NMR with complete ¹H decoupling.     

Proton line narrowing in solid-state nuclear magnetic resonance: new insights from windowed phase-modulated Lee-Goldburg sequence

We present here a bimodal Floquet analysis of the windowed phase-modulated Lee-Goldburg (wPMLG) sequence for homonuclear dipolar decoupling. One of the main criteria for an efficient homonuclear dipolar decoupling scheme is an effective z-rotation condition. This is brought about by the presence of radio-frequency imperfections in the pulse sequence together with a systematic manipulation of the wPMLG pulses. Additional improvement in the (1)H spectral resolution was obtained by a proper understanding of the off-resonance dependence of the wPMLG irradiation scheme based on bimodal Floquet theory. Numerical investigations further corroborate both theoretical and experimental findings. Theoretical analysis points to accidental degeneracies between the cycle time of the wPMLG sequence and the rotor period leading to the experimentally observed off-resonance dependence of the resolution. Two-dimensional (1)H-(1)H homonuclear single-quantum correlation spectra of model amino acids are also presented, highlighting the improved spectral resolution of wPMLG sequences.     

Characterization of dynamics of perdeuterated proteins by MAS solid-state NMR

We show in this communication that dynamic information for uniformly 2H,13C,15N isotopically enriched, crystalline proteins can be obtained by MAS solid-state NMR spectroscopy. The experiments make use of the deuterium quadrupolar tensor, which is the dominant interaction mechanism. Dynamic properties are accessed by measurement of the size of the quadrupolar coupling constant, Cq, and the value of the asymmetry parameter, eta, via evolution of the deuterium chemical shift, as well as by measurement of deuterium T1 relaxation times. Three-dimensional experiments are performed in order to obtain site-specific resolution. Due to proton dilution, no proton decoupling is required in the carbon evolution periods at MAS rotation frequencies of 10 kHz.     

Improved resolution in two-dimensional 1H NMR spectra of peptides by band-selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex. Complete assignment of the 1H-NMR spectra of the free and heparin-complexed peptide was achieved with the high resolution of the D-BASHD-TOCSY, ROESY and NOESY spectra. Characterization of the heparin-complexed peptide is of interest because of the ability of the peptide to neutralize the anticoagulant activity of heparin.     

Probing proton-proton proximities in the solid state: high-resolution two-dimensional 1H-1H double-quantum CRAMPS NMR spectroscopy

A new 1H DQ (double-quantum) CRAMPS (combined rotation and multiple-pulse sequence) solid-state nuclear magnetic resonance experiment incorporating DUMBO homonuclear 1H dipolar decoupling is presented. The major resolution enhancement enables DQ peaks corresponding to all 22 close (<3.5 A) proton-proton proximities in the dipeptide beta-AspAla to be observed. In particular, the DQ CRAMPS spectrum provides access to the alkyl region of the spectrum and yields a clear assignment of the two CH and two diastereotopic CH2 proton resonances.     

High resolution 1H detected 1H,13C correlation spectra in MAS solid-state NMR using deuterated proteins with selective 1H,2H isotopic labeling of methyl groups

MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme allows the acquisition of proton carbon correlation spectra with a resolution comparable to that in solution-state NMR experiments. We observe an increase in resolution by a factor of 10-15 compared to standard heteronuclear correlation experiments using PMLG for 1H,1H dipolar decoupling in the indirect dimension. At the same time, the full sensitivity of the proton-based experiment is retained. In comparison to the heteronuclear detected version of the experiment, a gain in sensitivity of a factor of approximately 4.7 is achieved.     

1H-1H dipolar couplings provide a unique probe of RNA backbone structure

A NMR method is described that permits simultaneous measurement of the geminal 2JH1H2 + 2DH1H2 splitting and the sum of the 1JCH1 + 1DCH1 + 1JCH2 + 1DCH2 couplings for methylene groups, where 2DH1H2 and 1DCH are residual dipolar couplings, occurring when molecules are weakly oriented relative to the magnetic field. By suppressing either the upfield or downfield half of the 1H-1H geminal doublet, the experiment yields improved resolution relative to regular two-dimensional 1H-13C correlation spectra, making it applicable to systems of considerable complexity. The method is demonstrated for measurement of all 2DH5'H5' couplings in a 24-nucleotide 13C-enriched RNA stem loop structure, weakly aligned in liquid crystalline Pf1. The method is equally applicable to methylene groups in 13C-labeled proteins and to natural abundance samples of smaller molecules.     

Boosting the Resolution of 1H NMR Spectra by Homonuclear Broadband Decoupling

Broadband homonuclear decoupling of proton spectra, that is, the collapse of all multiplets into singlets, has the potential of boosting the resolution of ¹H NMR spectra. Several methods have been described in the last 40 years to achieve this goal. Most of them can only be applied in the indirect dimension of multi‐dimensional NMR spectra or special data processing is necessary to yield decoupled 1D proton spectra. Recently, complete decoupling of proton spectra during acquisition has been introduced; this not only significantly reduced the experimental time to record these spectra, but also removed the need for any sophisticated processing schemes. Here we present an introduction and overview of the techniques and applications of broadband proton‐decoupled proton experiments.     

Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement

Membrane proteins play an important role in many biological functions. Solid-state NMR spectroscopy is uniquely suited for studying structure and dynamics of membrane proteins in a membranous environment. The major challenge to obtain high quality solid-state NMR spectra of membrane proteins is sensitivity, due to limited quantities of labeled high-molecular-weight proteins. Here we demonstrate the incorporation of paramagnetic metal (Cu(2+)) ions, through either EDTA or a chelator lipid, into membrane protein samples for rapid data collection under fast magic-angle spinning (MAS) and low power (1)H decoupling. Spectral sensitivity of DsbB (20 kDa), an integral membrane protein, more than doubles in the same experimental time due to (1)H T(1) relaxation enhancement by Cu(2+) ions, with DsbB native fold and active site intact. This technique can be implemented to acquire multidimensional solid-state NMR spectra for chemical shift assignments and structure elucidation of large membrane proteins with small sample quantities.     

NMR at 900 MHz

The very first high-resolution NMR spectra recorded at 900 MHz in July 2000 have demonstrated the benefits of increased magnetic field strength for studies of large biomolecules such as proteins and nucleic acids. Increased sensitivity and resolution for such molecules can only be observed in experiments that are optimized for transverse relaxation (TROSY). Substantial effects of magnetic alignment can easily be observed not only in paramagnetic proteins, but even in small molecules, such as chloroform. Such effects can be very useful for structural studies of biopolymers. The extreme resolution allows studies of very weak interactions in proteins. For instance, long-range H/D isotope effects are easily observed in H-N correlation experiments. The first systematic studies of relaxation properties of N-15 nuclei have been carried out for proteins at 500, 600, 800, and 900 MHz.     

Fullerene as a standard sample for adjusting the NMR spectral resolution in multiple-quantum magic-angle-spinning experiments.

A method using fullerene for adjusting the NMR spectral resolution for multiple quantum magic angle spinning (MQMAS) experiments is proposed. To observe its (13)C MAS signal, it is not necessary to apply (1)H decoupling, unavailable with single-resonance MQMAS probes. Although (13)C T(1) of fullerene is rather long, a recycle time of 5 s in shimming yields its signal with sufficiently high sensitivity if setting the appropriate Ernst angles corresponding to magnetic fields. It is demonstrated that so-adjusted high resolution is reflected in the (87)Rb MQMAS spectra of RbNO(3).     

Improved pulse sequences for pure exchange solid-state NMR spectroscopy

Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin.     

Unexpected effects of third-order cross-terms in heteronuclear spin systems under simultaneous radio-frequency irradiation and magic-angle spinning NMR

We recently noted [R. K. Harris, P. Hodgkinson, V. Zorin, J.-N. Dumez, B. Elena, L. Emsley, E. Salager, and R. Stein, Magn. Reson. Chem. 48, S103 (2010)] anomalous shifts in apparent (1)H chemical shifts in experiments using (1)H homonuclear decoupling sequences to acquire high-resolution (1)H NMR spectra for organic solids under magic-angle spinning (MAS). Analogous effects were also observed in numerical simulations of model (13)C,(1)H spin systems under homonuclear decoupling and involving large (13)C,(1)H dipolar couplings. While the heteronuclear coupling is generally assumed to be efficiently suppressed by sample spinning at the magic angle, we show that under conditions typically used in solid-state NMR, there is a significant third-order cross-term from this coupling under the conditions of simultaneous MAS and homonuclear decoupling for spins directly bonded to (1)H. This term, which is of the order of 100 Hz under typical conditions, explains the anomalous behaviour observed on both (1)H and (13)C spins, including the fast dephasing observed in (13)C{(1)H} heteronuclear spin-echo experiments under (1)H homonuclear decoupling. Strategies for minimising the impact of this effect are also discussed.     

Differential line broadening in MAS solid-state NMR due to dynamic interference

Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.     

A NMR method for the analysis of mixtures of alkanes with different deuterium substitutions

¹³C NMR at 125.76 MHz with ¹H and ²H decoupling, ²H NMR at 76.77 MHz with ¹H decoupling, and ¹H NMR at 500.14 MHz with ²H decoupling were employed as analytical tools to study the complex mixtures of deuterated ethanes resulting from the catalytic H–D exchange of normal ethane with gas-phase deuterium in the presence of a platinum foil. Reference samples consisting of 1:1 binary mixtures of pure normal ethane and ethane-d_n (n=1–6) were used to identify the peak positions in the ¹³C, ²H, and ¹H NMR spectra due to each individual isotopomer, and the effect of isotopic substitution on the chemical shifts was determined in each case. While the NMR of all three nuclei worked well for the identification of the individual components of the 1:1 standard mixtures, both ¹H and ²H NMR suffered from inadequate resolution when studying complex reaction mixtures because of the broadening of the lines due to ¹H–¹H (¹H NMR) and ²H–²H (²H NMR) couplings. ¹³C NMR was therefore determined to be the method of choice for the quantitative analysis of the reaction mixtures. Using the ¹³C NMR results, a correlation that takes into account the primary and secondary isotope substitution effects on chemical shifts was deduced. This equation was used for the identification of the individual components of the mixtures, and integration of the individual observed resonances was then employed for quantification of their composition. This study shows that ¹³C NMR with ¹H and ²H decoupling is a viable procedure for studying mixtures of deuterated ethanes. Furthermore, the additivity of the isotopic effects on chemical shifts and the transferability of the values obtained with ethane to other molecules makes this approach general for the analysis of other isotopomer mixtures.     

Real‐Time Band‐Selective Homonuclear Proton Decoupling for Improving Sensitivity and Resolution in Phase‐Sensitive J‐Resolved Spectroscopy

Real‐time band‐selective homonuclear ¹H decoupling during data acquisition of z‐filtered J‐resolved spectroscopy produces ¹H‐decoupled ¹H NMR spectra and leads to sensitivity enhancement and improved resolution, and thus aids the measurement of J couplings and residual dipolar couplings in crowded regions of ¹H NMR spectrum. High quality spectra from peptides, organic molecules, and also from enantiomers dissolved in weakly aligned chiral media are reported.     

Band‐selective excited ultrahigh resolution PSYCHE‐TOCSY: fast screening of organic molecules and complex mixtures

Precise assignments of ¹H atomic sites and establishment of their through‐bond COSY or TOCSY connectivity are crucial for molecular structural characterization by using ¹H NMR spectroscopy. However, this exercise is often hampered by signal overlap, primarily because of ¹H–¹H scalar coupling multiplets, even at typical high magnetic fields. The recent developments in homodecoupling strategies for effectively suppressing the coupling multiplets into nice singlets (pure‐shift), particularly, Morris's advanced broadband pure‐shift yielded by chirp excitation (PSYCHE) decoupling and ultrahigh resolution PSYCHE‐TOCSY schemes, have shown new possibilities for unambiguous structural elucidation of complex organic molecules. The superior broadband PSYCHE‐TOCSY exhibits enhanced performance over the earlier TOCSY methods, which however warrants prolonged experimental times due to the requirement of large number of dwell increments along the indirect dimension. Herein, we present fast and band‐selective analog of the broadband PSYCHE‐TOCSY, which is useful for analyzing complex organic molecules that exhibit characteristic yet crowded spectral regions. The simple pulse scheme relies on band‐selective excitation (BSE) followed by PSYCHE homodecoupling in the indirect dimension. The BSE‐PSYCHE‐TOCSY has been exemplified for Estradiol and a complex carbohydrate mixture comprised of six constituents of closely comparable molecular weights. The experimental times are greatly reduced viz., ~20 fold for Estradiol and ~10 fold for carbohydrate mixture, with respect to the broadband PSYCHE‐TOCSY. Furthermore, unlike the earlier homonuclear band‐selective decoupling, the BSE‐PSYCHE‐decoupling provides fully decoupled pure‐shift spectra for all the individual chemical sites within the excited band. The BSE‐PSYCHE‐TOCSY is expected to have significant potential for quick screening of complex organic molecules and mixtures at ultrahigh resolution. Copyright © 2015 John Wiley & Sons, Ltd.