Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo.
PD0325901 molecular weight Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.”
“In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148,
and F149) with alanine resulted in drastic changes in binding Entrectinib chemical structure affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.”
“Multiple-pass ultrahigh pressure homogenization (UHPH) was used for reducing microbial population of both indigenous spoilage microflora
in whole raw milk and a baroresistant pathogen (Staphylococcus aureus) inoculated in whole sterile milk to define pasteurization-like processing conditions. Response surface methodology was followed and Sclareol multiple response optimization of UHPH operating pressure (OP) (100, 175, 250 MPa) and number of passes (N) (15) was conducted through overlaid contour plot analysis. Increasing OP and N had a significant effect (P < 0.05) on microbial reduction of both spoilage microflora and Staph. aureus in milk. Optimized UHPH processes (five 202-MPa passes; four 232-MPa passes) defined a region where a 5-log10 reduction of total bacterial count of milk and a baroresistant pathogen are attainable, as a requisite parameter for establishing an alternative method of pasteurization. Multiple-pass UHPH optimized conditions might help in producing safe milk without the detrimental effects associated with thermal pasteurization.