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 (thG), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of thG-labelled dsDNA of different lengths. thG behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe thG-labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, thG was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, thG thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes.

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. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

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. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.

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. Even if the performance of the gold grid as SERS substrate exhibited a smaller enhancement factor than expected for silver, oxidized and reduced spectra of proteins (Сyt c, Hb and Mb) monolayers could be obtained and the characteristic redox dependent shifts of the marker bands ν19, ν4 and ν10 were seen. The easy modification protocol and the higher stability of the gold electrode towards oxidative currents are the advantages of the present spectroeletrochemical cell. Finally, FDTD simulations confirm that the roughness of the gold grid has an effect on the Raman enhancement of the adsorbed proteins.

Arg302 governs the pKa 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 Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.

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. A more benign approach relies on fluorescent nucleobases that can substitute their native counterparts with minimal perturbation, such as the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine (th G). To demonstrate the potency of 3HCnt and th G in deciphering interconversion mechanisms, we used the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into (+)/(-)PBS duplex, as a model system. When incorporated into the (-)PBS loop, the two probes were found to be highly sensitive to the individual steps both in the absence and the presence of a nucleic acid chaperone, providing the first complete mechanistic description of this critical process in HIV-1 replication. The combination of the two distinct probes appears to be instrumental for characterizing structural transitions of nucleic acids under various stimuli.

Mg2+ 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 Mg2+ However, the precise role of Mg2+ in this process and its impact on the structure and function of IM30 remains unknown. Here, we show that Mg2+ binds directly to IM30 with a binding affinity of ∼1 mm Mg2+ binding compacts the IM30 structure coupled with an increase in the thermodynamic stability of the proteins' secondary, tertiary, and quaternary structures. Furthermore, the structural alterations trigger IM30 double ring formation in vitro because of increased exposure of hydrophobic surface regions. However, in vivo Mg2+-triggered exposure of hydrophobic surface regions most likely modulates membrane binding and induces membrane fusion.

pKa 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 Escherichia coli (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. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+ and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.