During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.
Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.
During formation of HIV particles, the Gag polyproteins are thought to interact with Vpr proteins to promote their
encapsidation in the nascent particles. To directly visualize and monitor the formation of the Gag-Vpr complexes and
correlate their formation with Vpr oligomerization, we used two photon lifetime imaging microscopy (FLIM) and time
laps microscopy on HeLa cells expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the
biarsenical ReAsH labelling reagent. Using these fluorescent microscopy approaches, we found that Gag proteins interact
directly with Vpr proteins, which results in their transfer from the nuclear to the plasma membrane. Vpr oligomerization
was found critical for both its interaction with Gag proteins and its transfer to the plasma membrane. Moreover, mutations
in the C-terminal domain of Gag abolished the interaction with Vpr and its redistribution to the plasma membrane,
indicating that this domain was critical for interaction with Vpr. Taken together, these data show that the Gag polyproteins
interact through their C-terminal domain with Vpr oligomers, promoting the redistribution of the latter at the plasma
membrane and probably their incorporation into nascent viral particles.
In this work, we have investigated, by spectrally time-resolved pump-probe spectroscopy, the excited-state dynamics of the uv mutant of the green fluorescent protein (GFPuv). The gain dynamics of GFPuv is characterized by a mono-exponential behaviour and can be described by a simple model involving a photo-conversion between two form of the GFPuv chromophore. In order to obtain more information about the role played by the interaction between the chromophore and the proteic cage, we have performed spectrally time-resolved femtosecond experiment on synthetic GFP chromophore analogue. This study allows us to evidence the importance of chromophore-proteic cage interaction in the gain dynamics. Finally we investigated the excited-state dynamics of GFPuv fused with single chain antibody fragment (scFv). The subjacent idea is to use the dynamical photo-physical properties of GFPuv fused with scFv as folding reporter. By taking two scFvs, one is a well-folded antibody and one is a misfolded antibody, we have evidenced that the observed pump probe differential transmission spectra are affected by the presence of the misfolded antibody. This result shows that the tertiary structure of the protein can be modified by the presence of a misfolded scFv linked to the GFPuv.
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