FEMTOSTACK

The FEMTOSTACK project gathers inter-disciplinary know-how in ultrafast laser spectroscopy, theoretical chemistry and molecular biophysics and sets out to develop new experimental and simulation tools for the investigation of the structural dynamics of protein/nucleic acid complexes on femto- and millisecond time scales. The basic idea is to use ultrafast fluorescence quenching times due to intramolecular photo-oxidation as a hallmark for stable stacking interactions, like the ones afforded by pairs of tryptophan/guanine (Trp/G) aromatic residues. The system under study in the first research task is the nucleocapsid protein NCp7 from HIV, which is known to form such interactions, e.g. with the short primary binding sequences (+)/(-) PBS from the viral RNA. The same experimental approach will be applied to probe local DNA base dynamics when labelled with the fluorescent 2-aminopurine analogue (2-AP). The time-resolved fluorescence properties (quenching, spectral shifts, polarisation anisotropy) of 2-AP allows quantifying the structural heterogeneity (i.e stacked/unstacked with a near-by base) and how it is affected by binding of NCp7. The femtosecond time resolution together with appropriate mutations of the PBS sequences will shed new light on the nature of similar but apparently weaker interactions. As a matter of fact, the origin of the dynamic nature of binding is an open question since many years. These new experiments will thus help rationalizing the structural heterogeneity and dynamics, which remain out-of-reach by NMR spectroscopy. Using both Trp and 2-AP fluorescence as molecular reporters for local structural heterogeneity make femtosecond spectroscopy an interesting tool for the investigation of protein/nucleic acid interactions in general. In a separate development and in order to investigate slow millisecond structural transitions in a non-equilibrium protein/nucleic acid complex, we devise a new apparatus that will combine the low-volume consumption of microfluidic cells with the multi-channel capabilities and picosecond time resolution of a streak camera. The latter enables fluorescence lifetime imaging of up to 200 voxels along the microfluidic stream (multi-channel FLIM), thus allowing watching the structural heterogeneity of e.g. the 2-AP labels evolve as the complementary PBS sequences form "kissing complexes". This apparatus will then open a new avenue for the real-time observation of protein- or ligand induced changes in the local structure of oligonucleotides in general (e.g. TAT from HIV-1, CORE from HCV). The experimental work will be confronted with and complemented by molecular dynamics simulation, starting from partial knowledge borrowed from NMR structures of model systems, and combined with time-dependent density functional methods (TD-DFT) for evaluating the Trp/G electron transfer times. In a separate approach, recently developed methods combining molecular mechanics with continuum solvent models (MM/PBSA) will be used for evaluating the site-specific, nucleic-base-resolved contributions to free binding energies of NCp7 on the PBS sequences.