Size-Controlled Synthesis of Colloidal Gold Nanoparticles at Room Temperature Under the Influence of Glow Discharge

Highly dispersed colloidal gold (Au) nanoparticles were synthesized at room temperature using glow discharge plasma within only 5 min. The prepared Au colloids were characterized with UV–visible absorption spectra (UV–vis), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) equipped with an energy dispersion X-ray spectrometer (EDX). UV–vis, XPS and EDX results confirmed that Au3+ ions in HAuCl4 solution could be effectively reduced into the metallic state at room temperature with the glow discharge plasma. TEM images showed that Au nanoparticles were highly dispersed. The size of colloidal Au nanoparticles could be easily tuned in the nanometer range by adjusting the initial concentration of HAuCl4 solution. Moreover, the as-synthesized Au colloids (dav = 3.64 nm) exhibited good catalytic activity for glucose oxidation. The nucleation and growth of colloidal Au particles under the influence of the plasma was closely related with the high-energy electrons generated by glow discharge plasma

Insights into the role of the (α + β) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of Chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (α + β) insertion between the B7 β-strand and A7 α-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (α + β) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (α + β) domain, the thermal stability of the enzyme is substantially reduced, with an apparent Tm of 42.0 ± 1.0 °C, compared to the apparent Tm of 58.1 ± 1.0 °C of ChiA at pH 9.0. The specific activity of ChiAΔ(α + β) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered. © 2008 Elsevier B.V. All rights reserved

Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (β/α) 8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a nontwo-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, ΔG = 24.0 kcal/mol at 25°C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (β/α)8 barrel proteins, that their unfolding is a nontwo-state process, always involving at least one unfolding intermediate. © 2006 Wiley-Liss, Inc

Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (ΔH) and heat capacity change (ΔCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure. © 2004 Elsevier B.V. All rights reserved

Molecular to organismal chirality is induced by the conserved myosin 1D

International audienceThe emergence of asymmetry from an initially symmetrical state is a universal transition in nature. Living organisms show asymmetries at the molecular, cellular, tissular, and organismal level. However, whether and how multilevel asymmetries are related remains unclear. In this study, we show that Drosophila myosin 1D (Myo1D) and myosin 1C (Myo1C) are sufficient to generate de novo directional twisting of cells, single organs, or the whole body in opposite directions. Directionality lies in the myosins' motor domain and is swappable between Myo1D and Myo1C. In addition, Myo1D drives gliding of actin filaments in circular, counterclockwise paths in vitro. Altogether, our results reveal the molecular motor Myo1D as a chiral determinant that is sufficient to break symmetry at all biological scales through chiral interaction with the actin cytoskeleton

Thermal unfolding of human BRCA1 BRCT-domain variants

Missense mutations at the BRCT domain of human BRCA1 protein have been associated with an elevated risk for hereditary breast/ovarian cancer. They have been shown to affect the binding site and they have also been proposed to affect domain stability, severely hampering the protein's tumor suppressor function. In order to assess the impact of various such mutations upon the stability and the function of the BRCT domain, heat-induced denaturation has been employed to study the thermal unfolding of variants M1775R and R1699W, which have been linked with the disease, as well as of V1833M, which has been reported for patients with a family history. Calorimetric and circular dichroism results reveal that in pH 9.0, 5 mM borate buffer, 200 mM NaCl, analogously to wild type BRCT, all three variants undergo partial thermal unfolding to a denatured state, which retains most of the native's structural characteristics. With respect to wild-type BRCT, the mutation M1775R induces the most severe effects especially upon the thermostability, while R1699W also has a strong impact. On the other hand, the thermal unfolding of variant V1833M is only moderately affected relative to wild-type BRCT. Moreover, isothermal titration calorimetric measurements reveal that contrary to M1775R and R1699W variants, V1833M binds to BACH1 and CtIP phosphopeptides. © 2007 Elsevier B.V. All rights reserved