Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Effects of isovaleraldehyde on cell-sized lipid bilayer vesicles.

Membrane composition and components are intrinsic properties of a cell membrane. Any changes in lipid vesicle composition or any stimuli, such as heat, that affect molecular packing induce dynamic shape change. Dynamic shape changes allow the determination of structural organization changes upon a change in the membrane internal or external environment. In this study, we report how thermal stress can affect isovaleraldehyde (IVA) flavor compound-containing membranes. We revealed that (1) IVA-containing lipid vesicles are large and their increasing size results in increasing IVA/vesicle concentration; (2) IVA-containing lipid vesicles are less thermo-responsive and are affected by increasing IVA concentration; finally, we discussed (3) the molecular mechanisms behind membrane packing. We proposed that the characteristic of IVA-containing membranes could be used in evaluating drink quality. Our results would potentially contribute to the development of membrane technology and the progress in further understanding physiological processes, such as flavor sensation.