Whitfieldkastrup1859

Micro-oxygenation (Mox) is a common technique used to stabilize color and reduce harsh astringency in red wines. Here, we investigate the role of residual sugars, phenolics, SO2, and yeast on the oxidation of wine in three studies. In a Mox experiment, populations of yeasts emerged after the loss of SO2, and this was associated with sharp increases in oxygen consumption and acetaldehyde production. No acetaldehyde production was observed without the presence of yeast. In an oxygen saturation experiment, unfiltered wines, in particular those with residual sugar >3 g/L, consumed oxygen more quickly and produced more acetaldehyde than filtered wines. In a final experiment, the reincorporation of oxygen and glucose immediately after the completion of fermentation of an otherwise dry synthetic wine resulted in significant acetaldehyde production. These experiments highlighted the importance of yeast metabolism in determining a wine's response to Mox and suggested that the role of chemical oxidation to produce acetaldehyde during Mox may not be very important. It appears that control of microbial populations and residual sugar levels may be key to managing Mox treatments in winemaking, and production scale experiments should be conducted.Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π-cation, dipole-cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. YD23 research buy We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.Protein splicing is a self-catalyzed post-translational modification in which the intein enzyme excises itself from a precursor protein and ligates the flanking sequences to produce a mature protein. We report the solution structure of a 136-residue DnaX mini-intein enzyme derived from the cyanobacterium Spirulina platensis. This sequence adopts a well-defined globular structure and forms a horseshoe-shaped fold commonly found in the HINT (hedgehog intein) topology. Backbone dynamics and hydrogen exchange experiments revealed conserved motions on various time scales, which is proposed to be a characteristic of the intein fold. Interestingly, several dynamic motions were found in symmetrically equivalent positions within the protein structure, which might be a consequence of the symmetrical intein fold. In cell splicing activity showed that Spl DnaX mini-intein is a highly active enzyme. The precursor protein was not detected at any timepoint of the assay. Apart from the splicing reaction, catalytic cleavage at the N- and C-termini of the precursor protein was also observed. To determine the roles of the catalytic residues in splicing and cleavage reactions, all combinations of alanine mutations of these residues were generated and functionally characterized. This in-depth analysis revealed cooperativity between these catalytic residues, which suppresses the N- and C-terminal cleavage reactions and enhances the yield of the spliced product. Overall, this study provides a thorough structural, dynamic, and functional characterization of a new intein sequence and adds to the collection of these unique enzymes that have found tremendous applications in biochemistry and biotechnology.Hemodynamics plays a critical role in early diagnosis and investigating the growth mechanism of intracranial aneurysms (IAs), which usually induce hemorrhagic stroke, serious neurological diseases, and even death. We developed a transparent blood vessel-on-a-chip (VOC) device for magnetic resonance imaging (MRI) to provide characteristic flow fields of early IAs as the reference for early diagnosis. This VOC device takes advantage of the transparent property to clearly exhibit the internal structure and identify the needless air bubbles in the biomimetic fluid experiment, which significantly affects the MRI image quality. Furthermore, the device was miniaturized and easily assembled with arbitrary direction using a 3D-printed scaffold in a radiofrequency coil. Computational fluid dynamics (CFD) simulations of the flow field were greatly consistent with those data from MRI. Both internal flow and wall shear stress (WSS) exhibited very low levels during the IA growth, thus leading to the growth and rupture of IAs. PC-MRI images can also provide a reasonable basis for the early diagnosis of IAs. Therefore, we believed that this proposed VOC-based MR imaging technique has great potential for early diagnostic of intracranial aneurysms.Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself in a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called molecular darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves. We apply this technique to a simple dipeptide system, a ligand binding to T4 lysozyme L99A, and ligand binding to HIV integrase to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal and rotational/translational degrees of freedom in these systems.