Kevin J. Donovan

Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils

Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimers disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.

Ultrafast NMR T1 Relaxation Measurements: Probing Molecular Properties in Real Time

The longitudinal relaxation properties of NMR active nuclei carry useful information about the site-specific chemical environments and about the mobility of molecular fragments. Molecular mobility is in turn a key parameter reporting both on stable properties, such as size, as well as on dynamic ones, such as transient interactions and irreversible aggregation. In order to fully investigate the latter, a fast sampling of the relaxation parameters of transiently formed molecular species may be needed. Nevertheless, the acquisition of longitudinal relaxation data is typically slow, being limited by the requirement that the time for which the nucleus relaxes be varied incrementally until a complete build-up curve is generated. Recently, a number of single-shot-inversion-recovery methods have been developed capable of alleviating this need; still, these may be challenged by either spectral resolution restrictions or when coping with very fast relaxing nuclei. Here, we present a new experiment to measure the T1s of multiple nuclear spins that experience fast longitudinal relaxation, while retaining full high-resolution chemical shift information. Good agreement is observed between T1s measured with conventional means and T1s measured using the new technique. The method is applied to the real-time investigation of the reaction between D-xylose and sodium borate, which is in turn elucidated with the aid of ancillary ultrafast and conventional 2D TOCSY measurements.

Multiple Parallel 2D NMR Acquisitions in a Single Scan

Parallel receiving technologies have recently crossed the boundary separating magnetic resonance imaging from nuclear magnetic resonance (NMR) spectroscopy. In the latter case they promise significant time-saving advantages, by enabling the detection of multiple spectra simultaneously rather than in series. Moreover the full compatibility of parallel receiving with all other advances of contemporary NMR spectroscopy promises to open even further synergies in terms of speed and analytical capabilities. The present study shows one such instance, whereby the combination of parallel receiving multinuclear technologies is made with gradient-based spatial encoding methods, to yield multiple multidimensional NMR spectra in a single scan. The potential of this combination is demonstrated by the parallel acquisition of 2D H–H and H–S correlation spectra involving different S nuclei (F, P), within a single transient. Besides its potential conceptual message about what is nowadays within reach of NMR spectroscopy, the ensuing two-dimensional parallel ultrafast NMR spectroscopy (2D PUFSY) experiment carries new opportunities for high-throughput analyses, chemical kinetics, and fast experiments on metastable hyperpolarized solutions. Parallel receiving is an integral component of modern magnetic resonance; particularly in imaging applications where it can lead to substantial accelerating factors by scanning separate regions in space. The advent of multiple receivers is also beginning to influence NMR spectroscopy technologies; not by providing spatial multiplexing, but rather by enabling the simultaneous acquisition of two or more signals arising from different nuclei. Following the introduction of parallel NMR spectroscopy, it was shown that one of its main advantages results from its use to collect two or more different kinds of multidimensional correlation experiments, within the time duration that would normally entail to collect a single spectrum. This is the principle of parallel acquisition NMR spectroscopy (PANSY), which eventually evolved into more sophisticated pulse sequences capable of affording all the 2D correlation spectra necessary for a complete assignment of small molecules—within the timescale of the slowest experiment in the multiple set. These “parallel acquisition NMR, all-in-one combination of experimental applications” (PANACEA) strategies, have since been extended to systems of various heteronuclei and adapted to protein liquid-state and solid-state NMR experiments. While these multiple receiver techniques have demonstrated that substantial time savings are possible, they have still conformed to the classical means of indirect frequency encoding, whereby a series of independent scans are charged with encoding in a step-wise manner the evolution of the F1 indirect spectral domain. The incremented repetitions thus required to discretely sample the indirect time domain t1 implies that, even if sufficient sensitivity is available, sampling considerations associated with the slowest of all experiments still dictate the execution of all remaining 2D acquisitions. It was recently shown that sparse sampling coupled to non-Fourier processing techniques can alleviate this constraint, and break the Nyquist criteria without sacrifices in resolution or spectral bandwidth. Herein we present an alternative—and arguably ultimate—form of compressing multiple 2D experiments, involving their parallel implementation while following the spatially encoded protocol enabling the multiplexing of all the information involved in every indirect dimension, in a single scan. The spatiotemporal encoding principles underlying the acquisition of 2D NMR spectra/images in a single scan have been described elsewhere in detail, and hence they are only briefly described and within the context of the parallelized experiments presented here. “Ultrafast” NMR spectroscopy is based on endowing different z positions within a sample, with the different degrees of chemical shift evolution that would normally be associated with differing t1 values. If implemented in a one-to-one z–t1 fashion, this spatiotemporal encoding leads to a linear spatial winding of the magnetizations/coherences [Eq. (1)],

HyperBIRD: A Sensitivity-Enhanced Approach to Collecting Homonuclear-Decoupled Proton NMR Spectra†

Samples prepared following dissolution dynamic nuclear polarization (DNP) enable the detection of NMR spectra from low-γ nuclei with outstanding sensitivity, yet have limited use for the enhancement of abundant species like (1)H nuclei. Small- and intermediate-sized molecules, however, show strong heteronuclear cross-relaxation effects: spontaneous processes with an inherent isotopic selectivity, whereby only the (13)C-bonded protons receive a polarization enhancement. These effects are here combined with a recently developed method that delivers homonuclear-decoupled (1)H spectra in natural abundance samples based on heteronuclear couplings to these same, (13)C-bonded nuclei. This results in the HyperBIRD methodology; a single-shot combination of these two effects that can simultaneously simplify and resolve complex, congested (1)H NMR spectra with many overlapping spin multiplets, while achieving 50-100 times sensitivity enhancements over conventional thermal counterparts.

HyperSPASM NMR: a new approach to single-shot 2D correlations on DNP-enhanced samples.

Dissolution DNP experiments are limited to a single or at most a few scans, before the non-Boltzmann magnetization has been consumed. This makes it impractical to record 2D NMR data by conventional, t(1)-incremented schemes. Here a new approach termed HyperSPASM to establish 2D heteronuclear correlations in a single scan is reported, aimed at dealing with this kind of challenge. The HyperSPASM experiment relies on imposing an amplitude-modulation of the data by a single Δt(1) indirect-domain evolution time, and subsequently monitoring the imparted encoding on separate echo and anti-echo pathway signals within a single continuous acquisition. This is implemented via the use of alternating, switching, coherence selection gradients. As a result of these manipulations the phase imparted by a heteronucleus over its indirect domain evolution can be accurately extracted, and 2D data unambiguously reconstructed with a single-shot excitation. The nature of this sequence makes the resulting experiment particularly well suited for collecting indirectly-detected HSQC data on hyperpolarized samples. The potential of the ensuing HyperSPASM method is exemplified with natural-abundance hyperpolarized correlations on model systems.

Heteronuclear Cross‐Relaxation Effects in the NMR Spectroscopy of Hyperpolarized Targets

Dissolution dynamic nuclear polarization (DNP) enables high-sensitivity solution-phase NMR experiments on long-lived nuclear spin species such as (15)N and (13)C. This report explores certain features arising in solution-state (1)H NMR upon polarizing low-γ nuclear species. Following solid-state hyperpolarization of both (13)C and (1)H, solution-phase (1)H NMR experiments on dissolved samples revealed transient effects, whereby peaks arising from protons bonded to the naturally occurring (13)C nuclei appeared larger than the typically dominant (12)C-bonded (1)H resonances. This enhancement of the satellite peaks was examined in detail with respect to a variety of mechanisms that could potentially explain this observation. Both two- and three-spin phenomena active in the solid state could lead to this kind of effect; still, experimental observations revealed that the enhancement originates from (13)C→(1)H polarization-transfer processes active in the liquid state. Kinetic equations based on modified heteronuclear cross-relaxation models were examined, and found to well describe the distinct patterns of growth and decay shown by the (13)C-bound (1)H NMR satellite resonances. The dynamics of these novel cross-relaxation phenomena were determined, and their potential usefulness as tools for investigating hyperpolarized ensembles and for obtaining enhanced-sensitivity (1)H NMR traces was explored.

3D MAS NMR Experiment Utilizing Through-Space 15N–15N Correlations

We demonstrate a novel 3D NNC magic angle spinning NMR experiment that generates 15N-15N internuclear contacts in protein systems using an optimized 15N-15N proton assisted recoupling (PAR) mixing period and a 13C dimension for improved resolution. The optimized PAR condition permits the acquisition of high signal-to-noise 3D data that enables backbone chemical shift assignments using a strategy that is complementary to current schemes. The spectra can also provide distance constraints. The utility of the experiment is demonstrated on an M0Aβ1-42 fibril sample that yields high-quality data that is readily assigned and interpreted. The 3D NNC experiment therefore provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.

Proton Assisted Recoupling (PAR) in Peptides and Proteins

Proton-assisted recoupling (PAR) is examined by exploring optimal experimental conditions and magnetization transfer rates in a variety of biologically relevant nuclear spin-systems, including simple amino acids, model peptides, and two proteins-nanocrystalline protein G (GB1), and importantly amyloid beta 1-42 (M0Aβ1-42) fibrils. A selective PAR protocol, SUBPAR (setting up better proton assisted recoupling), is described to observe magnetization transfer in one-dimensional spectra, which minimizes experiment time (in comparison to two-dimensional experiments) and thereby enables an efficient assessment of optimal PAR conditions for a desired magnetization transfer. In the case of the peptide spin systems, experimental and simulated PAR data sets are compared on a semiquantitative level, thereby elucidating the interactions influencing PAR magnetization transfer and their manifestations in different spin transfer networks. Using the optimum Rabi frequencies determined by SUBPAR, PAR magnetization transfer trajectories (or buildup curves) were recorded and compared to simulated results for short peptides. PAR buildup curves were also recorded for M0Aβ1-42 and examined conjointly with a recent structural model. The majority of salient cross-peak intensities observed in the M0Aβ1-42 PAR spectra are well-modeled with a simple biexponential equation, although the fitting parameters do not show any strong correlation to internuclear distances. Nevertheless, these parameters provide a wealth of invaluable semiquantitative structural constraints for the M0Aβ1-42. The results presented here offer a complete protocol for recording PAR 13C-13C correlation spectra with high-efficiency and using the resulting information in protein structural studies.