No abstract

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, β2-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of β2-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process.

␤2-microglobulin (␤2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified ␤2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G ␤2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G ␤2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the ␤2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type ␤2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G ␤2m mutant.Among the amyloidogenic proteins that have been most frequently studied over the last years, there is ␤ 2 -microglobulin (␤2m) 4 that is responsible for dialysis-related amyloidosis.␤2m, the non-polymorphic light chain of class I major histocompatibility complex, is a small protein that converts into fibrils without the necessity of any chemical modification. Fibril formation occurs in vitro, and most probably also in vivo, through the intact protein. This allows extending experimental conclusions from in vitro studies to the natural process. More importantly, ␤2m recapitulates exquisitely the paradigm of the partially unfolded intermediate, bridging the native fold and the fibrillar conformation, as traditionally invoked to explain the conformational transition from the native fold to the amyloid. Therefore, a detailed characterization of all intermediate states occurring along the folding pathway is of great importance to understand the process of fibril formation. A kinetic scheme of ␤2m refolding entailing two fast steps, burst phase and fast phase, prior to reaching a slow conversion phase from the intermediate to the native state, was first reported by Chiti et al. (1). This long-lived ␤2m refolding intermediate, termed I 2 or I T by different authors, has long been recognized as an effective fibril-competent species, formerly regarded as an ensemble of species (1) and later as a single species (2-4). It has been shown that I T contains a non-native trans peptide bond between His-31 and Pro-32 that slowly converts into cis conformation during the final refolding step (3). The isomerization occurs with minor rearrangements of the protein toward the native structure from an already native-lik...

Down syndrome (DS) is caused by the presence of a supernumerary copy of the human chromosome 21 (Hsa21) and is the most frequent genetic cause of intellectual disability (ID). Key traits of DS are the distinctive facies and cognitive impairment. We conducted for the first time an analysis of the Nuclear Magnetic Resonance (NMR)-detectable part of the metabolome in plasma and urine samples, studying 67 subjects with DS and 29 normal subjects as controls selected among DS siblings. Multivariate analysis of the NMR metabolomic profiles showed a clear discrimination (up to of 80% accuracy) between the DS and the control groups. The univariate analysis of plasma and urine revealed a significant alteration for some interesting metabolites. Remarkably, most of the altered concentrations were consistent with the 3:2 gene dosage model, suggesting effects caused by the presence of three copies of Hsa21 rather than two: DS/normal ratio in plasma was 1.23 (pyruvate), 1.47 (succinate), 1.39 (fumarate), 1.33 (lactate), 1.4 (formate). Several significantly altered metabolites are produced at the beginning or during the Krebs cycle. Accounting for sex, age and fasting state did not significantly affect the main result of both multivariate and univariate analysis.