Pressure pushes tRNA into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNA, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling.

Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation.

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways.

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (M) and HCV NS3/4A protease.

Protein unfolded states populated at high and ambient pressure are similarly compact.

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature.

The Deep Sea Osmolyte Trimethylamine N-Oxide and Macromolecular Crowders Rescue the Antiparallel Conformation of the Human Telomeric G-Quadruplex from Urea and Pressure Stress.

Organisms are thriving in the deep sea at pressures up to the 1 kbar level, which imposes severe stress on the conformational dynamics and stability of their biomolecules. The impact of osmolytes and macromolecular crowders, mimicking intracellular conditions, on the effect of pressure on the conformational dynamics of a human telomeric G-quadruplex (G4) DNA is explored in this study employing single-molecule Förster resonance energy transfer (FRET) experiments. In neat buffer, pressurization favors the parallel/hybrid state of the G4-DNA over the antiparallel conformation at ≈400 bar, finally leading to unfolding beyond 1000 bar.

Temperature and pressure limits of guanosine monophosphate self-assemblies.

Guanosine monophosphate, among the nucleotides, has the unique property to self-associate and form nanoscale cylinders consisting of hydrogen-bonded G-quartet disks, which are stacked on top of one another. Such self-assemblies describe not only the basic structural motif of G-quadruplexes formed by, e.g.

Multiple helical conformations of the helix-turn-helix region revealed by NOE-restrained MD simulations of tryptophan aporepressor, TrpR.

The nature of flexibility in the helix-turn-helix region of E. coli trp aporepressor has been unexplained for many years. The original ensemble of nuclear magnetic resonance (NMR structures showed apparent disorder, but chemical shift and relaxation measurements indicated a helical region.

Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein.

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry.

1.2 Å resolution crystal structure of Escherichia coli WrbA holoprotein.

The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported.

Biphasic kinetic behavior of E. coli WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase.

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds.

Environment-dependent long-range structural distortion in a temperature-sensitive point mutant.

Extensive environment-dependent rearrangement of the helix-turn-helix DNA recognition region and adjacent L-tryptophan binding pocket is reported in the crystal structure of dimeric E. coli trp aporepressor with point mutation Leu75Phe. In one of two subunits, the eight residues immediately C-terminal to the mutation are shifted forward in helical register by three positions, and the five following residues form an extrahelical loop accommodating the register shift.