Thienoguanosine, a unique non-perturbing reporter for investigating rotational dynamics of DNA duplexes and their complexes with proteins.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine (G), a fluorescent analogue of guanosine, overcomes all these limitations.

Kinetics of protein-assisted nucleic acid interconversion monitored by transient time resolved fluorescence in microfluidic droplets.

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments.

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study.

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway.

Surface-enhanced resonance Raman spectroscopy of heme proteins on a gold grid electrode.

The combination of surface-enhanced resonance Raman spectroscopy (SERRS) and electrochemistry is an ideal tool to study the redox process of the heme proteins and is often performed on silver electrodes. In this manuscript, we present an approach using a microstructured gold surface that serves as the electrochemical working electrode, and at the same time, acts as SERS active substrate. The cell requires a micromolar concentration of sample at the electrode surface.

Arg302 governs the pK of Glu325 in LacY.

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H, using the free energy released from downhill translocation of H to drive accumulation of galactosides against a concentration gradient. In neutrophilic , internal pH is kept at ∼7.

Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures.

Nucleic acids are characterized by a variety of dynamically interconverting structures that play a major role in transcriptional and translational regulation as well as recombination and repair. To monitor these interconversions, Förster resonance energy transfer (FRET)-based techniques can be used, but require two fluorophores that are typically large and can alter the DNA/RNA structure and protein binding. Additionally, events that do not alter the donor/acceptor distance and/or angular relationship are frequently left undetected.

Mg binding triggers rearrangement of the IM30 ring structure, resulting in augmented exposure of hydrophobic surfaces competent for membrane binding.

The "inner membrane-associated protein of 30 kDa" (IM30), also known as "vesicle-inducing protein in plastids 1" (Vipp1), is found in the majority of photosynthetic organisms that use oxygen as an energy source, and its occurrence appears to be coupled to the existence of thylakoid membranes in cyanobacteria and chloroplasts. IM30 is most likely involved in thylakoid membrane biogenesis and/or maintenance, and has recently been shown to function as a membrane fusion protein in presence of Mg However, the precise role of Mg in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg binds directly to IM30 with a binding affinity of ∼1 mm Mg binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures.

pK of Glu325 in LacY.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H across the cytoplasmic membrane of (galactoside/H symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding.