Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein

The 72 kDa heat shock protein (HSP72) is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV) nucleocapsid protein (N), a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins

Large-Eddy Simulations with remeshed Vortex methods: An assessment and calibration of subgrid-scale models

This study assesses various subgrid-scale models within the framework of Large Eddy Simulation (LES) using a remeshed Vortex method (RVM). RVM is a semi-Lagrangian method discretizing the vorticity-velocity Navier–Stokes equations that has proven to be a stable and less dissipative alternative to more classical Eulerian methods. The subgrid-scale models are first tested on the well-known Taylor–Green Vortex case at Re=5000. Notably, the Variational Multiscale (VMS) variant of the Smagorinsky model and the Spectral Vanishing Viscosity (SVV) approaches emerge as the best-suited to the RVM, as they add diffusion to only the smallest resolved vorticity scales. Then, a stochastic uncertainty quantification analysis is conducted for both selected models, and the model coefficients are calibrated against direct numerical simulation. These coefficients are then applied to additional cases (different regimes, grid resolutions and test cases), showing the robustness of the calibration within the RVM-LES framework

ATPase-Defective Derivatives of Escherichia coli DnaK That Behave Differently with Respect to ATP-Induced Conformational Change and Peptide Release

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a ΔdnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell