We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range 13C-13C correlation spectra and a third based on the identification of intermolecular interactions in 13C-15N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3), that efficient 13C-13C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The 13C-13C experiments are corroborated by 15N-13C correlation spectrum obtained from a mixed [15N,12C]/[14N,13C] sample which directly quantifies interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular 15N-13C constraints present in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total the experiments provide 111 intermolecular 13C-13C and 15N-13C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.

A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ؎ 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-␤ structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.amyloid aggregation ͉ amyloid oligomers ͉ two-color coincidence spectroscopy ͉ PI3-SH3 domain ͉ neurodegenerative diseases

The SH3 domain of the PI3 kinase (PI3-SH3 or PI3K-SH3) readily aggregates into fibrils in vitro and has served as an important model system to investigate the molecular properties and mechanism of formation of amyloid fibrils. We describe the molecular conformation of PI3-SH3 in amyloid fibril form as revealed by magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy. The MAS NMR spectra of these fibrils display excellent resolution, with narrow 13C and 15N line widths, representing a high degree of structural order and the absence of extensive molecular motion for the majority of the polypeptide chain. We have identified the spin-systems of 82 of the 86 residues in the protein, and obtained sequential resonance assignments for 75 of them. Chemical shift analysis indicates that the protein subunits making up the fibril adopt a compact conformation consisting of four well-defined β-sheet regions and four random-coil elements with varying degrees of local dynamics or disorder. The backbone conformation of PI3-SH3 in fibril form differs significantly from that of the native state of the protein, both in secondary structure and in the location of dynamic or disordered segments. The site-specific MAS NMR analysis of PI3-SH3 fibrils we report here is compared with previously published mechanistic and structural data, resulting in a detailed interpretation of the factors that mediate fibril formation by PI3-SH3 and allowing us to propose a possible model of the core structure of the fibrils. Our results confirm the structural similarities between PI3-SH3 fibrils and amyloids directly related to degenerative or infectious diseases.

Proteins with a high propensity to aggregate can be largely prevented from doing so with surprisingly small changes to their primary structure. By using a combination of rational design and quantitative measurements of aggregation rates, we show that adding a single charge in specific "gatekeeper" regions is sufficient to change the timescale for amyloid fibril growth from minutes to weeks, thereby dramatically reducing the efficiency of this process.

Solid-state NMR is a powerful technique for the investigation of complex biological systems such as membrane proteins and amyloid fibrils. In magic-angle spinning (MAS) NMR, structural information is obtained via the reintroduction of anisotropic interactions.[1] In particular, a large number of homonuclear dipolar recoupling schemes have been developed and applied to record correlation spectra and measure internuclear distances in peptides and proteins.[1] While many recoupling techniques efficiently transfer polarization between directly bonded 13 C nuclei, their effectiveness can be reduced significantly when distant 13 C spins with weak dipolar couplings are involved. This limitation is generally imposed both by the experimental constraints that must be maintained during long mixing periods and by the inherent complexities of multiple-spin systems such as dipolar truncation, [2,3] that is the attenuation of weak dipolar couplings by stronger dipolar couplings in the recoupled dipolar Hamiltonian. As a consequence, spin diffusion techniques, [4][5][6][7][8] which circumvent some of these limitations, [9] have been widely utilized to estimate long-range homonuclear structural constraints in protein solid-state NMR studies. [10,11] In this communication we present an experimental approach that provides highly sensitive long-range correlations between aliphatic 13 C nuclei through the combination of isotope dilution and efficient polarization transfer via a band selective radio frequency dipolar recoupling (BASE RFDR) scheme. We demonstrate this method with a sample of PI3-SH3 (the SH3 domain of the p85α subunit of phosphatidylinositol 3 kinase) in amyloid fibril form.The 13 C alternating labeling scheme devised by LeMaster and Kushlan,[12] and introduced to solid-state NMR by Hong,[13] which employs 2-13 C 1 or 1,3-13 C 2 glycerol as the carbon source, results in amino acids 13 C labeled at approximately every other carbon position ("odd/even" labeling).