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SABRE is a nuclear spin hyperpolarization technique based on the reversible association of a substrate molecule and para-hydrogen (p-H2) to a metal complex. During the lifetime of such a complex, generally fractions of a second, the spin order of p-H2 is transferred to the nuclear spins of the substrate molecule via a transient scalar coupling network, resulting in strongly enhanced NMR signals. This technique is generally applied at relatively high concentrations (mM), in large excess of substrate with respect to metal complex. Dilution of substrate ligands below stoichiometry results in progressive decrease of signal enhancement, which precludes the direct application of SABRE to the NMR analysis of low concentration (μM) solutions. Here, we show that the efficiency of SABRE at low substrate concentrations can be restored by addition of a suitable coordinating ligand to the solution. The proposed method allowed NMR detection below 1 μM in a single scan.

Nuclear magnetic resonance is often the technique of choice in chemical analysis because of its sensitivity to molecular structure, quantitative character, and straightforward sample preparation. However, determination of trace analytes in complex mixtures is generally limited by low sensitivity and extensive signal overlap. Here, we present an approach for continuous hyperpolarization at high magnetic field that is based on signal amplification by reversible exchange (SABRE) and can be straightforwardly incorporated in multidimensional NMR experiments. This method was implemented in a 2D correlation experiment that allows detection and quantification of analytes at nanomolar concentration in complex solutions.

Amyloid is the result of an anomalous protein and peptide aggregation, leading to the formation of insoluble fibril deposits. At present, 18 human diseases have been associated with amyloid deposits-e.g., Alzheimer's disease and Prion-transmissible Spongiform Encephalopathies. The molecular structure of amyloid is to a large extent unknown, because of lack of high-resolution structural information within the amyloid state. However, from other experimental data it has been established that amyloid fibrils predominantly consist of ␤-strands arranged perpendicular to the fibril axis. Identification of residues involved in these secondary structural elements is therefore of vital importance to rationally designing appropriate inhibitors. We have designed a hydrogen͞ deuterium exchange NMR experiment that can be applied on mature amyloid to enable identification of the residues located inside the fibril core. Using a highly amyloidogenic peptide, corresponding to residues 25-35 within the Alzheimer A␤(1-43) peptide, we could establish that residues 28 -35 constitute the amyloid core, with residues 31 and 32 being the most protected. In addition, quantitative values for the solvent accessibility for each involved residue could be obtained. Based on our data, two models of peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors.A myloid diseases have in common an abnormal folding of normally soluble proteins resulting in the formation of extracellular amyloid deposits (1). Examples are Alzheimer's Disease, Prion-transmissible Spongiform Encephalopathies, and Familial Amyloidotic Polyneuropathy (2). Through selfassembly these proteins produce regular fibrillar structures possessing a predominant ␤-sheet conformation (3). Structural information on the core of fibrils has mainly been elucidated from fiber-diffraction studies (4), mass-spectrometry (5), and solid-state NMR spectroscopy (6), resulting in a general picture consistent with parallel or antiparallel ␤-strands, placed perpendicularly to the fibril axis. However, a detailed structure of a fibril at the atomic level is still lacking. In this respect, more structural information-e.g., which specific amino acids make up the amyloid-forming core-becomes crucially important.In this article, we describe a NMR approach to determining the sequence-specific structural elements in the fibril stage of amyloid forming proteins. The method relies on the partial solvent protection of hydrogen-bonded amide protons connecting the ␤-strands throughout the length of the fibril. In aqueous solutions it is expected that amide protons located on the exterior of the fibril are more accessible to solvent and therefore experience a higher hydrogen exchange rate than amide protons buried within the fibril interior. Studies using mass spectrometry, in combination with deuterium exchange, have already pointed to this possibility (7). However, mass spectrometry does not allow spe...