The three-dimensional structural analysis of DNA undecamer 5'd(C1G2C3A4C5X6C7A8C9G10C11)3', 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a modified abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran] has been performed using NOESY, DQFCOSY, TOCSY, and 31P-1H HSQC-TOCSY spectra in relation with molecular dynamics simulations. A total of 249 distances and 224 dihedral angles were used for construction. The optimal distances were calculated using the complete relaxation matrix method from hybrid matrices which were built with the experimental NOE intensities and additional data derived from either standard A- or B-DNA. Six independent refined structures starting from canonical A- and B-DNA were determined on the basis of the NMR data, and all converged to a single family with average rms deviations below 0.6 A and final NOE Rx factors of 0.055 +/- 0.03. A satisfactory agreement was obtained between measured NOE intensities and those resulting from full relaxation matrix calculations. A single intrahelical form of right-handed DNA duplex is observed; the aromatic base of residue T17 opposite the abasic site is stacked inside the helix. No clear correlation was detected between the C5 and C7 residues, excluding their proximity and the looping out of the abasic site. The abasic site induces a kink of about 30 degrees in the DNA duplex. This kink allows the formation of a bifurcated hydrogen bond between the amino protons of C5 and the O4 oxygen of T17. A detailed analysis of the final structures and their comparison with previous studies of abasic site lesions are described.

Three variants of human ␤ 2 -microglobulin (␤ 2 -m) were compared with wild-type protein. For two variants, namely the mutant R3A␤ 2 -m and the form devoid of the N-terminal tripeptide (⌬N3␤ 2 -m), a reduced unfolding free energy was measured compared with wild-type ␤ 2 -m, whereas an increased stability was observed for the mutant H31Y␤ 2 -m. The solution structure could be determined by 1 H NMR spectroscopy and restrained modeling only for R3A␤ 2 -m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Y␤ 2 -m and ⌬N3␤ 2 -m. Precipitation and unfolding were observed over time periods shorter than 4 -6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for ␤ 2 -m fibrillogenesis. The mechanism is consistent with the previous and present results on ␤ 2 -m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.Over the last several years, an overwhelming number of reports have addressed the phenomenon of amyloidogenesis. The interest in the subject stems not only from the social relevance of amyloid pathologies such as Alzheimer's disease, or spongiform encephalopathy, or the various systemic amyloidoses, but also from the general implications in the issue of protein folding.Amyloidoses have been recognized as conformational diseases that arise from the conversion of globular proteins into insoluble fibrillar aggregates (1). Despite the diversity of the involved proteins, amyloid fibrils exhibit a common structure known as cross-␤ structure, which appears to be a particularly stable, generic protein fold, accessible to many polypeptide chains under specific conditions in vitro and in vivo (2). The amyloid deposition of ␤ 2 -microglobulin (␤ 2 -m), 1 the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I), is associated to dialysis-related amyloidosis (3). The disease is the result of long term hemodialysis in individuals with chronic renal failure, a widespread pathology with high social costs that are further increased by the inevitable dialysis-related amyloidosis complication. Recently ankylosing spondylitis has also been proposed to originate from ␤ 2 -m deposition (4). We determined the solution structure of isolated ␤ 2 -m by NMR spectroscopy (5) and showed that the most important rearrangements of the protein, with respect to its structure in MHC-I, were observed for strands D and E, interstrand loop D-E, and strand A, including the N-terminal segment. We stated that these modifications can be conside...

Abnormal aggregation of beta-amyloid (Abeta) peptides into toxic aggregates has been identified as a key event in Alzheimer's disease (AD). Inhibition of this process has thus emerged as a major therapeutic track against AD. The present work describes the synthesis and in vitro study of a novel class of inhibitors. Two copies of Abeta-binding motifs (either curcumin or the KLVFFA peptide) are clicked via copper(I)-mediated azide-alkyne cycloaddition on a constrained cyclopeptide scaffold designed to interfere with Abeta aggregation. Our conjugates strongly inhibit amyloid fibril formation from Abeta(40) at low inhibitor to Abeta molar ratios (e.g., 0.02:1 in the case of the KLVFFA conjugate) at which Abeta-binding motifs alone are fully inactive (thioflavin T assays and atomic force microscopy observation). This work highlights the value of combining Abeta-recognition domains with a steric hindrance-inducing scaffold for preventing amyloid fibril formation.