Solvent Removal Induces a Reversible ß-to-α Switch in Oligomeric Aß Peptide.

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 Aß peptide experiences a remarkable conformational switch from ß to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aß 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 Aß peptide in amyloid oligomers that could bear relevance for Aß's interactions with cellular structures of low polarity.

Structure and biomedical applications of amyloid oligomer nanoparticles.

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.

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra.

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 ß1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

Chemical shift correlation at high MAS frequencies employing low-power symmetry-based mixing schemes.

An approach for conveniently implementing low-power CN ( n ) (ν) and RN ( n ) (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 (15)N-(13)C dipolar and (13)C-(13)C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.

Broadband 15N-13C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies.

An approach for generating efficient NR(vS, vk)(n) symmetry-based dual channel RF pulse schemes for gamma-encoded broadband (15)N-(13)C 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 (15)N-(13)C chemical shift correlation experiments. Employing such (13)C-(15)N 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.

Numerical design of RN(n)(nu) symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies.

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.

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CN(n)(nu) symmetry-based RF pulse schemes.

The CN(n)(nu) 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(n)(nu) 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.

Design of high-power, broadband 180 degrees pulses and mixing sequences for fast MAS solid state chemical shift correlation NMR spectroscopy.

An approach for the design of high-power, broadband 180 degrees pulses and mixing sequences for generating dipolar and scalar coupling mediated (13)C-(13)C chemical shift correlation spectra of isotopically labelled biological systems at fast magic-angle spinning frequencies without (1)H 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.

Heteronuclear J cross-polarisation in liquids using amplitude and phase modulated mixing sequences.

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.

Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes.

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.

Broadband homonuclear chemical shift correlation at high MAS frequencies: a study of tanh/tan adiabatic RF pulse schemes without 1H decoupling during mixing.

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.

Tailoring broadband inversion pulses for MAS solid state NMR.

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.

Solid state NMR at high magic angle spinning frequencies: dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes.

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.

Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy.

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).

TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs.

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.

Adiabatic heteronuclear decoupling in rotating solids.

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.