Differential Scanning Fluorimetry provides high throughput data on silk protein transitions

Here we present a set of measurements using Differential Scanning Fluorimetry (DSF) as an inexpensive, high throughput screening method to investigate the folding of silk protein molecules as they abandon their first native melt conformation, dehydrate and denature into their final solid filament conformation. Our first data and analyses comparing silks from spiders, mulberry and wild silkworms as well as reconstituted ‘silk’ fibroin show that DSF can provide valuable insights into details of silk denaturation processes that might be active during spinning. We conclude that this technique and technology offers a powerful and novel tool to analyse silk protein transitions in detail by allowing many changes to the silk solutions to be tested rapidly with microliter scale sample sizes. Such transition mechanisms will lead to important generic insights into the folding patterns not only of silks but also of other fibrous protein (bio)polymers

A Novel Tetrameric PilZ Domain Structure from Xanthomonads

PilZ domain is one of the key receptors for the newly discovered secondary messenger molecule cyclic di-GMP (c-di-GMP). To date, several monomeric PilZ domain proteins have been identified. Some exhibit strong c-di-GMP binding activity, while others have barely detectable c-di-GMP binding activity and require an accessory protein such as FimX to indirectly respond to the c-di-GMP signal. We now report a novel tetrameric PilZ domain structure of XCC6012 from the plant pathogen Xanthomonas campestris pv. campestris (Xcc). It is one of the four PilZ domain proteins essential for Xcc pathogenicity. Although the monomer adopts a structure similar to those of the PilZ domains with very weak c-di-GMP binding activity, it is nevertheless interrupted in the middle by two extra long helices. Four XCC6012 proteins are thus self-assembled into a tetramer via the extra heptad repeat α3 helices to form a parallel four-stranded coiled-coil, which is further enclosed by two sets of inclined α2 and α4 helices. We further generated a series of XCC6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer. Discovery of this new PilZ domain architecture increases the complexity of c-di-GMP-mediated regulation

Free Cysteine Modulates the Conformation of Human C/EBP Homologous Protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid l-cysteine. Addition of l-cysteine caused a significant dose-dependent decrease in the protein helicity – dropping from 69.1% to 23.8% in the presence of 1 mM of l-cysteine – and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8∶1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free l-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of l-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress

Étude cinétique de la synthèse des carbocyanines. I. Aminolyse des sels de ß-ethylthiovinyl-2 cycloammoniums

Summary. — Salts of β-ethylthiovinyl cycloammoniums are generated, as stable intermediates, during the synthesis of carbocyanines starting from orthoesters and quaternary salts with activated methyl groups. In order to set up a reaction scheme of this synthesis, reactivity of these intermediates towards nucleophiles was investigated. Therefore, kinetical parameters of the reaction of 2(β-ethylthiovinyl) 1,3,3-trimethyl indoleninium iodide (trans form) with a series of anilines were determined. The reaction is looked at in acetonitrile at 25 °C. The observed rate constant (κapp.) follows the equation : [formule] pointing out the double part played by the anilines (A).Guillaume A., Bruylants Albert. Étude cinétique de la synthèse des carbocyanines. I. Aminolyse des sels de ß-ethylthiovinyl-2 cycloammoniums. In: Bulletin de la Classe des sciences, tome 61, 1975. pp. 901-927

Étude cinétique de la synthèse des carbocyanines. III. Action des bases méthyléniques sur les sels de β-éthylthiovinyl-2 cycloammoniums et catalyse par les anions

The third paper of this series is dealing with the kinetics of the carbocyanine synthesis. The reaction of free methylene bases, like 2-methy-lene 1,3,3-trimethylindolenine and its chlorinated derivative, with 2-(β-ethyl-thiovinyl) 1,3,3 trimethylindoleninium salts was found to be first order with respect to the free base. Tertiary bases were found to be ineffective as catalysts, whereas anions (CI-, Br-, I-) are weak catalysts. On the contrary, when the methylene base is made in situ — for instance by pyridine reacting with 1,2,3,3 tetramethylindoleninium salts — the same anions are good catalysts. Competition between anions and pyridine for the pyridinium cation generated in a pre-equilibrium was evidenced.Guillaume A., Bruylants Albert. Étude cinétique de la synthèse des carbocyanines. III. Action des bases méthyléniques sur les sels de β-éthylthiovinyl-2 cycloammoniums et catalyse par les anions . In: Bulletin de la Classe des sciences, tome 62, 1976. pp. 177-198