Dynamics and calcium association to the N-terminal regulatory domain of human cardiac troponin C: a multiscale computational study.

Troponin C (TnC) is an important regulatory molecule in cardiomyocytes. Calcium binding to site II in TnC initiates a series of molecular events that result in muscle contraction. The most direct change upon Ca(2+) binding is an opening motion of the molecule that exposes a hydrophobic patch on the surface allowing for Troponin I to bind. Molecular dynamics simulations were used to elucidate the dynamics of this crucial protein in three different states: apo, Ca(2+)-bound, and Ca(2+)-TnI-bound. Dynamics between the states are compared, and the Ca(2+)-bound system is investigated for opening motions. On the basis of the simulations, NMR chemical shifts and order parameters are calculated and compared with experimental observables. Agreement indicates that the simulations sample the relevant dynamics of the system. Brownian dynamics simulations are used to investigate the calcium association of TnC. We find that calcium binding gives rise to correlative motions involving the EF hand and collective motions conducive of formation of the TnI-binding interface. We furthermore indicate the essential role of electrostatic steering in facilitating diffusion-limited binding of Ca(2+)

Structural and biochemical characterization of human Schlafen 5

The Schlafen family belongs to the interferon-stimulated genes and its members are involved in cell cycle regulation, T cell quiescence, inhibition of viral replication, DNA-repair and tRNA processing. Here, we present the cryo-EM structure of full-length human Schlafen 5 (SLFN5) and the high-resolution crystal structure of the highly conserved N-terminal core domain. We show that the core domain does not resemble an ATPase-like fold and neither binds nor hydrolyzes ATP. SLFN5 binds tRNA as well as single- and double-stranded DNA, suggesting a potential role in transcriptional regulation. Unlike rat Slfn13 or human SLFN11, human SLFN5 did not cleave tRNA. Based on the structure, we identified two residues in proximity to the zinc finger motif that decreased DNA binding when mutated. These results indicate that Schlafen proteins have divergent enzymatic functions and provide a structural platform for future biochemical and genetic studies

On the ion coupling mechanism of the MATE transporter ClbM

Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli, which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter.</p

Quasi-variational coupled-cluster theory: Performance of perturbative treatments of connected triple excitations

Quasi-variational coupled-cluster methods are applied to a selection of diatomic molecules. The potential energy curves, spectroscopic constants, and size consistency errors are calculated and compared to those obtained from both single- and multi-reference methods. The effects of connected triple excitations are introduced with either the standard perturbative (T) formulation, or in the renormalised form, and its symmetrised approximation. It is found that the renormalised ansatz is significantly superior to the standard formulation when describing bond breaking and that in most circumstances, the computationally simpler symmetrisation gives nearly identical results

Dynamics of crowded vesicle: local and global responses to membrane composition

The bacterial cell envelope is composed of a mixture of different lipids and proteins, making it an inherently complex organelle. The interactions between integral membrane proteins and lipids are crucial for their respective spatial localization within bacterial cells. We have employed microsecond timescale coarse-grained molecular dynamics simulations of vesicles of varying sizes and with a range of protein and lipid compositions, and used novel approaches to measure both local and global system dynamics, the latter based on spherical harmonics analysis. Our results suggest that both hydrophobic mismatch, enhanced by embedded membrane proteins, and curvature based sorting, due to different modes of undulation, may drive assembly in vesicular systems. Interestingly, the modes of undulation of the vesicles were found to be altered by the specific protein and lipid composition of the vesicle. Strikingly, lipid dynamics were shown to be coupled to proteins up to 6 nm from their surface, a substantially larger distance than has previously been observed, resulting in multi-layered annular rings enriched with particular types of phospholipid. Such large protein-lipid complexes may provide a mechanism for long-range communication. Given the complexity of bacterial membranes, our results suggest that subtle changes in lipid composition may have major implications for lipid and protein sorting under a curvature-based membrane-sorting model

Linker length affects photostability of protein-targeted sensor of cellular microviscosity.

Viscosity sensitive fluorophores termed 'molecular rotors' represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications