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Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aβ peptide experiences a remarkable conformational switch from β to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aβ fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aβ peptide in amyloid oligomers that could bear relevance for Aβ's interactions with cellular structures of low polarity.

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC'C and 3D C'NCA with sequential (13)C acquisitions, 3D NHH and 3D NC'H with sequential (1)H acquisitions and 3D CANH and 3D C'NH with broadband (13)C-(15)N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

An approach for generating efficient $$ {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} $$ symmetry-based dual channel RF pulse schemes for γ-encoded broadband 15N–13C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and 15N–13C chemical shift correlation experiments. Employing such 13C–15N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.

An approach for the efficient implementation of RN(n)(nu) symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN(n)(nu) symmetry involve the repeated application of the pulse sandwich {R(phi)R(-phi)}, corresponding to a propagator U(RF) = exp(-i4phiI(z)), where phi = pinu/N and R is typically a pulse that rotates the nuclear spins through 180 degrees about the x-axis. In this study, broadband, phase-modulated 180 degrees pulses of constant amplitude were employed as the initial 'R' element and the phase-modulation profile of this 'R' element was numerically optimised for generating RN(n)(nu) symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving 13C-13C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here.

The CN ν class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic “C” element corresponding to an RF cycle with unity propagator. In this study, the design of CN ν symmetry-based RF pulse sequences for achieving 13C–13C double-quantum dipolar recoupling and through bond scalar coupling mediated 13C–13C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic “C” elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the “C” element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here.

The design of mixing sequences for heteronuclear J cross-polarisation in the liquid state has been examined employing supercycles of amplitude/phase modulated RF pulses. The Fourier coefficients defining the modulation profiles of the pulses were optimised numerically so as to achieve efficient magnetisation transfer within the desired range of resonance offsets. A variety of supercycles, pulsewidths and RF field strengths were considered in implementing heteronuclear anisotropic and isotropic mixing sequences. The coherence transfer characteristics of the sequences obtained were evaluated by numerical simulations. The experimental performances of the sequences were tested by measurements carried out on a moderate sized protein at 750 MHz. The results presented demonstrate that the approach adopted in this study can be employed effectively to tailor, as per the experimental requirements and constraints, the RF-field modulation profiles of the pulses constituting the mixing scheme for generating heteronuclear J cross-polarisation sequences.

Heteronuclear dipolar decoupling in MAS solid state NMR studies of biological systems has been investigated at moderate spinning speeds. It is shown that the efficacy of C N n ν symmetry-based decoupling schemes with tanh/tan adiabatic pulses can be significantly improved by optimising the amplitude and frequency modulation characteristics of the inversion pulses employed. The decoupling performance of the symmetry-based schemes is compared with that of the commonly used TPPM technique.

The thiolate complexes Zn(15]aneN(4))(S-CH2-C6H5)]ClO4 (1) (15aneN(4) = 1,4,8,12-tetraazacyclopentadecane) and Zn(i-14]aneN(4))(S-CH2-C6H5)ICIO4 (2) (i-14aneN(4) = 1, 4,7, 11 -tetraazacyclotetradecane) have been reacted with carbon disulfide. The trithiocarbonate complexes Zn(15]aneN(4))S-C(S)-S-CH2-C6H5]CIO4 1a, monoclinic, space group P2(1)/n, Z = 8, a = 13,2338(1) angstrom, b = 12.9251(2) angstrom, c = 30.1669(4) angstrom, beta = 101.463(1)degrees, V = 5057.1(1) angstrom(3) and Zn(i14]aneN(4))S-C(S)-S-CH2-C6H5]CIO4 2a, orthorhombic, space group P2(1)2(1)2(1), Z = 8, a = 9.9936(1) angstrom, b 22.1261(4) angstrom, c = 22.3192(4) angstrom, V = 4935.2(l) angstrom(3) were obtained. The reaction of 1 with CS2 is second order with a rate constant of k = (57.6 +/- 2.4) x 10(-3) m(-1.)s(-1) at 25 degrees C. The experimentally determined Eyring activation barrier is Delta H-exp(double dagger) = 65.3 +/- 0.7 kJ(.)mol(-1) (Delta S-exp(double dagger) = -49.9 +/- 2.5 J(.)mol(-1.)K(-1)) and a free energy of activation of Delta G* = 80.2 +/- 1.5 kJ(.)mol(-1) at 25 degrees C. To discriminate between an associative and a dissociative mechanism the barriers for both processes were calculated using density functional theory at the C-PCM(B98/G3MP2Large)//B3LYP/ 6-311+G(d) level. The associative mechanism is clearly favored with a difference in free energies of activation of delta Delta G(double dagger) approximate to 80kJ(.)mol(-1). Its calculated barrier Delta G(theor)(double dagger) = 114.3 kJ(.)mol(-1) is in reasonable agreement with the experimental value. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006).

It is shown that it is possible to carry out with only a moderate RF field strength of ⩽40 kHz, even at high magic angle spinning frequencies, broadband homonuclear double-quantum filtered 13 C chemical shift correlation and double-quantum solid state NMR studies of biological systems via the zero-quantum dipolar recoupling sequence RFDR.

It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).

We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.

The performance of symmetry-based adiabatic RF pulse schemes for 1 H decoupling at moderate magic angle spinning (MAS) frequencies has been evaluated via numerical simulations and experimentally compared with other decoupling techniques. Our studies reveal that in MAS solid state NMR studies of polycrystalline specimens, in contrast to symmetry-based RF pulse schemes involving rectangular RF pulses, adiabatic decoupling sequences can be effectively implemented to make use of all the available decoupling RF field strength and to eliminate the deleterious effects of H 1 inhomogeneity and resonance offset. Symmetry-based adiabatic decoupling sequences lead to improved decoupling performance compared to a continuous train of adiabatic inversion pulses that are phase cycled as in solution state NMR studies.

A MAS solid state NMR approach for achieving efficient scalar coupling mediated through-bond (13)C chemical shift correlations of the aliphatic carbons in uniformly labelled peptides/proteins is described. The method involves the application of a continuous train of adiabatic inversion pulses, as in the adiabatic TOCSY experiments carried out in solution state NMR studies. While rotor synchronised application of adiabatic inversion pulses leads to dipolar correlations, it is shown here via numerical simulations and experimental measurements that asynchronous application of adiabatic pulses can facilitate the mapping of through-bond connectivities. The method employs a suitable phasing scheme for generating the desired isotropic mixing Hamiltonian and requires moderate (13)C RF field strength only.

In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.

RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.

A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly ((13)C, (15)N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear (13)C-(15)N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power (1)H decoupling in experiments requiring long t (1) acquisition times.

The growth of protein crystals is preceded by an induction period during which the protein solution prepares itself for crystallization. We have measured the length of the induction period for lysozyme as a function of the temperature for a solution of 16.9 mg/mL at pH 4.5 with 5% NaCl as the precipitating agent. The results are described with classical nucleation theory. The number of molecules in the critical nucleus n* turns out to be quite small: n* = 3-9. Such a small aggregate can hardly be considered as crystalline, as is assumed in the classical theory. By taking into account the anisotropy of the interaction between lysozyme molecules, we show that small nuclei, with n

With a view to obtain 13C chemical shift correlation spectra of uniformly labelled peptides/proteins at high magnetic fields and high magic angle spinning frequencies (ωr/2π ≤ 20 kHz), the efficacy of RFDR with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements employing different adiabatic pulse phasing schemes, shapes and durations. It is demonstrated that homonuclear dipolar recoupling with superior performance under resonance offset and H 1 inhomogeneity effects and without strong dependence on the 13C chemical shift differences can be achieved with adiabatic pulses. It is shown that 13C chemical shift correlation spectra in the entire range of carbon chemical shifts can be obtained efficiently with short adiabatic inversion pulses. In situations where correlation spectra of only the aliphatic region are required, the possibility for minimising the interference between the recoupling and decoupling RF fields with long adiabatic pulses, at low recoupling power levels and without compromising the broadband RFDR characteristics, is also indicated.

It is shown that it is possible to effectively execute RFDR experiments with adiabatic inversion pulses and obtain resonance offset compensation that is superior to what can be achieved by conventional rectangular pulses. Employing 40-μs tanh/tan adiabatic pulses at a power level of ∼38 kHz and a spinning speed of 12 kHz it is demonstrated that the range of resonance offset compensation achieved is sufficient to generate, via a single experiment, homonuclear chemical shift correlation spectra in the entire 13C chemical shift range in peptides/proteins at the currently available field strengths.

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3–6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model 15N-13C spin systems, (13C′,15N) Aib-(15N) Aib-NH2 (Aib = α-aminoisobutyric acid) and (1-13C,15N) glycine.

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimer's and Parkinson's. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging.

In a dipolar-coupled spin-1/2 network of the type 15 N1– 13 C– 15 N2, an assessment of the sensitivity of the N → C and C → N TEDOR sideband intensities to the Euler angles defining the orientation of the two heteronuclear dipolar vectors in the 13 C and 15 N chemical shift (CS) tensor principal axes system has been carried out via numerical calculations. The results clearly indicate the potential of TEDOR MAS NMR spectroscopy for the characterization of the CS tensor orientation in the molecular frame. The efficacy of the method has been experimentally illustrated by TEDOR studies on a polycrystalline sample of [1,3— 15 N 2 , 2— 13 C]uracil, which is one of the four bases in RNA.

An approach to the determination of the orientation of the carbonyl chemical shift (CS) tensor in a 13C′-15N-1H dipolar coupled spin network is proposed. The method involves the measurement of the Euler angles of the 13C′-15N and 15N-1H dipolar vectors in the 13C′ CS tensor principal axes system, respectively, via a 13C-15N REDOR experiment and by a 2D relayed anisotropy correlation of the 13C′ CSA (ω2) and 15N-1H dipolar interaction (ω1). Via numerical simulations the sensitivity of the ω1 cross sections of the 2D spectrum to the Euler angles of the 15N-1H bond vector in the 13C′ CSA frame is shown. Employing the procedure outlined in this work, we have determined the orientation of the 13C′ CS tensor in the peptide plane of the dipeptide AibAib-NH2 (Aib = α-aminoisobutyric acid). The Euler angles are found to be (χCN, ψCN) = (34° ± 2°, 88° ± 2° ) and (χNH, ψNH) = (90° ± 10°, 80° ± 10° ). From the measured Euler angles it is seen that the σ33 and σ22 components of the 13C′ CS tensor approximately lie in the peptide plane.

The response of a spin (1/2) ensemble, at thermal equilibrium and experiencing chemical shift anisotropy (CSA), to the application of adiabatic inversion pulses has been studied under magic-angle spinning (MAS). Numerical simulations and experimental studies on such systems, carried out under slow spinning conditions, show that the response to adiabatic inversion pulses has much more favorable characteristics than the response to conventional rectangular pulses. We have also explored the possibilities of employing adiabatic 180 degrees pulses as dephasing pulses in rotational-echo double-resonance (REDOR) experiments. Our results show that it is indeed possible to employ such adiabatic inversion pulses conveniently in REDOR experiments to eliminate resonance offset and H(1) inhomogeneity effects which may arise from the usage of conventional rectangular 180 degrees pulses. Copyright 2000 Academic Press.

As part of our studies on the characterization of 15N chemical shift anisotropy (CSA) via magic angle spinning (MAS) NMR spectroscopy, we have investigated via numerical simulations the sensitivity of two different REDOR experimental protocols to the angles defining the orientation of the 15N-13C' bond vector in the principal axis system of the 15N CSA tensor of the amide nitrogen in a peptide bond. Additionally, employing polycrystalline samples of 15N and 13C', 15N-labeled acetanilide, we have obtained, in a first study of this type, the orientation of the 15N CSA tensor in the molecular frame by orienting the tensor with respect to the 15N-3C' and 15N-1H dipolar vectors via 15N-13C' REDOR and 15N-1H dipolar-shift MAS experiments, respectively.

An approach to the determination of the 2-(13)C' chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S1-I-S2 consisting of directly bonded heteronuclear spins, we have carried out numerical simulations to assess the sensitivity of I-S REDOR spinning sidebands to the Euler angles defining the orientation of the I-S1 and I-S2 dipolar vectors in the I spin CS tensor principal axes system. Our investigations clearly demonstrate the potential of I-S REDOR studies in IS1S2 systems for obtaining with high reliability and accuracy the I spin chemical shift tensor orientation in the molecular frame spanned by the two internuclear vectors I-S1 and I-S2. The significant contribution to the observed REDOR sideband intensities from anti-phase operator terms which are present at the start of the data acquisition is illustrated. The procedure for the recording and analysis of the I-S REDOR spectra in IS1S2 systems is presented and the measurement of the 2-(13)C' CS tensor orientation in a polycrystalline sample of [1,3-(15)N2, 2-(13)C] uracil, which is one of the four bases in RNA, is experimentally demonstrated.

Aggregation of b-amyloid (Ab) peptide into oligomers and protofibrils is a hallmark of Alzheimer s disease (AD). Increasing evidence shows that the primary insult in AD is caused by oligomeric species that impair the ordered function of synaptic networks. Consistent with this view, oligomers were shown to affect synaptic plasticity, and they impair the long-term potentiation (LTP) in living brain tissues, a widely used model system of brain memory functions. Using solidstate NMR spectroscopy, we here determined the residuespecific molecular conformation of a highly synaptotoxic bamyloid oligomer structure. Our measurements reveal a stable N-terminal b strand that controls the partitioning between oligomer and protofibril formation, whereas targeting the peptide N-terminus ameliorates Ab-dependent neuronal dysfunctions. The presently investigated, chemically well-defined Ab oligomers faithfully reproduce the hallmark characteristics of AD-related oligomers. Living hippocampal brain slices were exposed to different Ab conformers (Figure 1A), and a series of tetanic electrical stimuli were applied to evoke a longlasting increase of the synaptic transmission, termed LTP. Oligomers, but not freshly dissolved, that is, primarily monomeric, Ab peptide or fibrils, reduce the LTP response and therefore disturb the brain memory functions within these tissue samples (Figure 1B). A similar oligomer-specificity is seen with cultured primary neurons, which present a significant oligomer-dependent decrease ( 40%) of their

An approach for conveniently implementing low-power CN ν and RN ν symmetry-based band-selective mixing sequences for generating homo- and heteronuclear chemical shift correlation NMR spectra of low γ nuclei in biological solids is demonstrated. Efficient magnetisation transfer characteristics are achieved by selecting appropriate symmetries requiring the application of basic RF elements of relatively long duration and numerically tailoring the RF field modulation profile of the basic element. The efficacy of the approach is experimentally shown by the acquisition of 15N–13C dipolar and 13C–13C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.

An approach for the design of high-power, broadband 180° pulses and mixing sequences for generating dipolar and scalar coupling mediated 13C–13C chemical shift correlation spectra of isotopically labelled biological systems at fast magic-angle spinning frequencies without 1H decoupling during mixing is presented. Considering RF field strengths in the range of 100–120 kHz, as typically available in MAS probes employed at high spinning speeds, and limited B 1 field inhomogeneities, the Fourier coefficients defining the phase modulation profile of the RF pulses were optimised numerically to obtain broadband inversion and refocussing pulses and mixing sequences. Experimental measurements were carried out to assess the performance characteristics of the mixing sequences reported here.

We have explored the design of broadband scalar coupling mediated (13)C-(13)C and cross-relaxation suppressed (1)H-(1)H TOCSY sequences employing phase/amplitude modulated inversion pulses. Considering a variety of supercycles, pulsewidths and a RF field strength of 10 kHz, the Fourier coefficients defining the amplitude and phase modulation profiles of the 180 degrees pulses were optimised numerically so as to obtain efficient magnetisation transfer within the desired range of resonance offsets. The coherence transfer characteristics of the mixing schemes were assessed via numerical simulations and experimental measurements and were compared with commonly used sequences based on rectangular RF pulses. The efficacies of the clean (1)H-(1)H TOCSY sequences were also examined via numerical simulations for application to weakly oriented systems and sequences with efficient, broadband and clean dipolar transfer characteristics were identified. In general, the amplitude and phase modulated TOCSY sequences presented here have moderately better performance characteristics than the sequences currently employed in biomolecular NMR spectroscopy.

At high magic angle spinning (MAS) frequencies the potential of tanh/tan adiabatic RF pulse schemes for 13C chemical shift correlation without 1H decoupling during mixing has been evaluated. It is shown via numerical simulations that a continuous train of adiabatic 13C inversion pulses applied at high RF field strengths leads to efficient broadband heteronuclear decoupling. It is demonstrated that this can be exploited effectively for generating through-bond and through-space, including double-quantum, correlation spectra of biological systems at high magnetic fields and spinning speeds with no 1H decoupling applied during the mixing period. Experiments carried out on a polycrystalline sample of histidine clearly suggest that an improved signal to noise ratio can be realised by eliminating 1H decoupling during mixing.

A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H(1) inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR.

The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H(1) inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.

The efficacy of RN(nu) (n) symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RN(nu) (n) sequences generally employ basic inversion elements that correspond to a net 180 degrees rotation about the rotating frame x-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RN(nu) (n) schemes employing adiabatic pulses. Such an approach was successfully used for obtaining (13)C chemical shift correlation spectra of a uniformly labelled sample of (CUG)(97) - a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the (13)C sugar carbon chemical shifts suggests, in agreement with our recent (15)N MAS-NMR studies, that this RNA adopts an Alpha-helical conformation.

With a view to obtain (13)C chemical shift correlation spectra of uniformly labelled peptides/proteins at high magnetic fields and high magic angle spinning frequencies (omega(r)/2pi/= 20 kHz), the efficacy of RFDR with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements employing different adiabatic pulse phasing schemes, shapes and durations. It is demonstrated that homonuclear dipolar recoupling with superior performance under resonance offset and H(1) inhomogeneity effects and without strong dependence on the (13)C chemical shift differences can be achieved with adiabatic pulses. It is shown that (13)C chemical shift correlation spectra in the entire range of carbon chemical shifts can be obtained efficiently with short adiabatic inversion pulses. In situations where correlation spectra of only the aliphatic region are required, the possibility for minimising the interference between the recoupling and decoupling RF fields with long adiabatic pulses, at low recoupling power levels and without compromising the broadband RFDR characteristics, is also indicated.

It is shown that it is possible to effectively execute RFDR experiments with adiabatic inversion pulses and obtain resonance offset compensation that is superior to what can be achieved by conventional rectangular pulses. Employing 40-micros tanh/tan adiabatic pulses at a power level of approximately 38 kHz and a spinning speed of 12 kHz it is demonstrated that the range of resonance offset compensation achieved is sufficient to generate, via a single experiment, homonuclear chemical shift correlation spectra in the entire (13)C chemical shift range in peptides/proteins at the currently available field strengths.

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3-6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model 15N-13C spin systems, (13C',15N) Aib-(15N) Aib-NH2 (Aib = alpha-aminoisobutyric acid) and (1-13C,15N) glycine.

The potential of heteronuclear MAS NMR spectroscopy for the characterization of (15)N chemical shift (CS) tensors in multiply labeled systems has been illustrated, in one of the first studies of this type, by a measurement of the chemical shift tensor magnitude and orientation in the molecular frame for the two (15)N sites of uracil. Employing polycrystalline samples of (15)N(2) and 2-(13)C, (15)N(2)-labeled uracil, we have measured, via (15)N-(13)C REDOR and (15)N-(1)H dipolar-shift experiments, the polar and azimuthal angles (theta;, psi) of orientation of the (15)N-(13)C and (15)N-(1)H dipolar vectors in the (15)N CS tensor frame. The (theta;(NC), psi(NC)) angles are determined to be (92 +/- 10 degrees, 100 +/- 5 degrees ) and (132 +/- 3 degrees, 88 +/- 10 degrees ) for the N1 and N3 sites, respectively. Similarly, (theta;(NH), psi(NH)) are found to be (15 +/- 5 degrees, -80 +/- 10 degrees ) and (15 +/- 5 degrees, 90 +/- 10 degrees ) for the N1 and N3 sites, respectively. These results obtained based only on MAS NMR measurements have been compared with the data reported in the literature.

Using numerical optimization procedures it is shown that it is possible to design composite 180° RF pulses for MAS NMR spectroscopy by explicitly taking into account the variation of the resonance offset of each crystallite during the application of the RF pulses. When using composite RF pulses in experiments such as TOSS, where the delays between the RF pulses have to be critically adjusted, an optimization of these delays can lead to the desired performance characteristics.