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The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ1-40 and Aβ1-42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ1-42 compared to that of Aβ1-40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ1-40 and Aβ1-42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ1-40 and Aβ1-42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings (³JHNHa, ³JC'C', ³JC'Ha, ¹JHαCα, ²JNCα, and 1JNCα) recorded for Aβ1-40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ɸ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ1-42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process. [ABSTRACT FROM AUTHOR]

Provided that care is taken in adjusting the WATERGATE element of a H-N TROSY-HSQC experiment, such that neither the water magnetization nor the H protons are inverted by its final 180° pulse, J couplings can be measured directly from splittings in the H dimension of the spectrum. With band-selective H decoupling, very high N resolution can be achieved. A complete set of J values, ranging from 3.4 to 10.1 Hz was measured for the 56-residue third domain of IgG-binding protein G (GB3). Using the H-N-C-H dihedral angles extracted from a RDC-refined structure of GB3, J values predicted by a previously parameterized Karplus equation agree to within a root-mean-square deviation (rmsd) of 0.37 Hz with the experimental data. Values measured for the Alzheimer's implicated Aβ peptide fit to within an rmsd of 0.45 Hz to random coil J values. [ABSTRACT FROM AUTHOR]

Abstract: Spectral density mapping at multiple NMR field strengths is probably the best method to describe the dynamics behavior of a protein in solution through the analysis of 15N heteronuclear relaxation parameters. Nevertheless, such analysis is scarcely reported in the literature, probably because this method is excessively demanding in spectrometer measuring time. Indeed, when using n different magnetic fields, the discrete sampling of the spectral density function with 2n +1 points needs the measurement of 3n 15N heteronuclear relaxation measurements (n R 1, n R 2, and n 15N{1H} NOEs), assuming the validity of the high frequency approximation. Based on further approximations, we proposed a new strategy that allows us to describe the spectral density with n +2 points, with the measurement of a total of n +2 heteronuclear relaxation parameters. Applied to the dynamics analysis of the protein C12A-p8MTCP1 at five different NMR fields, this method allowed us to divide by nearly a factor 2 the total measuring time, without altering further results obtained by the Lipari–Szabo “model-free” analysis of the resulting spectral densities. [Copyright &y& Elsevier]

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Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the #916;mbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. [ABSTRACT FROM AUTHOR]