Several lines of evidence indicate that pre-fibrillar assemblies of amyloid- (A)polypeptides

Angles between two interatomic vectors are measured for structure elucidation in solution nuclear magnetic resonance (NMR). The angles can be determined directly by using the effects of dipole-dipole cross-correlated relaxation of double-quantum and zero-quantum coherences. The measured rates can be directly related to the angular geometry without need for calibration of a Karplus-type curve, as is the case for scalar coupling measurements, and depend only on the rotational correlation time of the molecule as an empirical parameter. This makes the determination of torsional angles independent from the measurement of coupling constants. The two interatomic vectors can in principle be arbitrarily far apart. The method was demonstrated on the measurement of the peptide backbone angle psi in the protein rhodniin, which is difficult to determine in solution by NMR spectroscopy.

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.

Slim peaks: Using a perdeuterated protein recrystallized from a 10:90 H2O:D2O mixture in magic‐angle spinning (MAS) solid‐state NMR spectroscopy experiments gives small 1H line widths at moderate spinning frequencies without application of homonuclear decoupling. This labeling strategy opens new perspectives for assignment of large protein spin systems.

The three-dimensional structure of the chemotactic peptide Nformyl-L-Met-L-Leu-L-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 13 C-15 N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly 13 C, 15 N-and 15 N-labeled samples. The peptide's structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Relatively large proteins in solution, spun in NMR rotors for solid samples at typical ultracentrifugation speeds, sediment at the rotor wall. The sedimented proteins provide high-quality solid-state-like NMR spectra suitable for structural investigation. The proteins fully revert to the native solution state when spinning is stopped, allowing one to study them in both conditions. Transiently sedimented proteins can be considered a novel phase as far as NMR is concerned. NMR of transiently sedimented molecules under fast magic angle spinning has the advantage of overcoming protein size limitations of solution NMR without the need of sample crystallization/precipitation required by solid-state NMR.ferritin | magic angle spinning NMR | sedimentation | gravity | high molecular weight "W e built a new ultracentrifuge which permitted the study of solutions in fields of force of up to about 100,000 times gravity…it is possible to cover the entire sector of the colloids down to the smallest particle sizes, and even…reach…the substances of high molecular weight, such as haemoglobin, protein, starch, etc.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58␣ subunit of bovine phosphatidyl-inositol-3-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as ␣-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to ␣-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.protein misfolding ͉ protein aggregation ͉ protein evolution T he spontaneous conversion of soluble proteins or protein fragments into aggregates and amyloid fibrils is a challenging problem in biological and medical sciences. An increasing body of evidence supports the anomalous misassembly of proteins into insoluble deposits as the fundamental cause behind a growing number of debilitating human disorders, such as Alzheimer's disease and Parkinson's disease, type II diabetes, and the transmissible spongiform encephalopathies (1-4). Important clues to understand the molecular basis of amyloid diseases and, more generally, the biological significance of protein aggregation have emerged recently from observations made in proteins unrelated to human disease, which have been found to convert in vitro into aggregates with structural and cytotoxic properties indistinguishable from those exhibited by amyloid assemblies associated with pathological conditions (5-14).The Src homology 3 (SH3) domain of the p58␣ subunit of phosphatidyl-inositol-3Ј-kinase (PI3-SH3) is one of the best characterized examples of a small globular protein unrelated to any known pathological condition that can form amyloid fibrils in vitro (5,15,16). Aggregated species obtained from this protein have been found to be cytotoxic when added to cell cultures (13). This observation suggests that the protein aggregates underlying different human disorders could show similar mechanisms of cytotoxicity and more generally that during evolution nature had to develop strategies to avoid protein misassembly to preserve the viability of liv...

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