A zirconium-based metal–organic framework sensitized by thioflavin-T for sensitive photoelectrochemical detection of C-reactive protein

Herein, a photoelectrochemical (PEC) assay was proposed for sensitive detection of C-reactive protein (CRP) based on zirconium-based metal-organic framework (PCN-777) as the photoelectric material and thioflavine-T (Th-T) as the sensitizer.

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants.

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.

An Engineered PrPsc-like Molecule from the Chimera of Mammalian Prion Protein and Yeast Ure2p Prion-inducing Domain

Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the “protein-only” hypothesis. In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD). Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content β-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

The folding of β2-microglobulin (β2-m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I2, which slowly converts into the native fold, N. Here we show that the partially folded species I2can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I2conformation is populated to ∼14 ± 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I2conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of β2-min vivoinvolving the association of the preformed fibrils with the fraction of I2existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of β2-m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating β2-m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.