Biomolecular Structural Heterogeneity and Dynamics studied by ultrafast spectroscopy

Natalia Grytsyk, Stefan Haacke, Jérémie Léonard

Biomolecules bear a natural flexibility, which leads to the observation that they can exist in multiple conformations, like polymers. Structural heterogeneity and dynamics are an integral part of the biomolecules functional versatility and interactions. Characterizing structural heterogeneity in terms of relative abundance of different conformers, and resolving structural dynamics would allow a mechanistic understanding of biomolecular interactions at the molecular scale. This in turn would enable the rational design of innovative therapeutic strategies targeting specifically these interactions.

Our approach is to apply Time-Resolved Fluorescence (TRF) spectroscopy for the investigation of heterogeneous biomolecular systems tagged with different fluorescent labels, as reporters for the local conformations and transitions between the conformational states.

Probing structural heterogeneity by TRF

As a model system we have been investigating short DNA hairpins, labeled with 2 amino-purine, a synthetic fluorescence analog of adenine. Molecular dynamics simulation (coll. With R. Stote, IGBMC) is used to model the distribtuion of conformers that coexist in solution. In different conformers 2AP experiences a different environment leading to a different fluorescence lifetime. Hence a distribution of conformers should yield a distribution of fluorescence lifetime, in ensemble measurement. We use TRF to characterize the fluorescence decay kinetics of 2AP in these DNA hairpins and we showed that the observed distribution of lifetimes can be exploited to validate the conformational heterogeneity predicted by MD simulations.

“Ultrafast Site-specific Fluorescence Quenching of 2-Aminopurine in a DNA hairpin studied by femtosecond down-conversion”, Thomas Gelot, Patricia Tourón-Touceda, Olivier Crégut, Jérémie Léonard and Stefan Haacke, J. Phys. Chem. A, 116, 2819-2825, 2012.

« Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy », Karine Voltz, Jérémie Léonard, Patricia Tourón Touceda, Jamie Conyard, Ziyad Chaker, Annick Dejaegere, Julien Godet, Yves Mély, Stefan Haacke, Roland H. Stote, Nucleic Acids Research, 2016, 44, 3408-3419.

Funding: ANR FemtoSTACK (2011-14), ANR Pico2 (2015-2019)

Figure: left: distribution of fluorescence lifetime obtained by analyzing the fluorescence decay kinetics of 21AP in 3 distinct DNA haipins. Right The distribution of fluorescence lifetimes are rationalized in terms of distribution of conformers the structure and relative population of which may be modeled by molecular dynamics simulations. From K. Voltz et al. Nucleic Acids Research, 44, 3408-3419, (2016).

Towards Probing structural dynamics by TRF

To resolved structural dynamics, we are exploring a new experimental approach enabling the investigation of out-of-equilibrium biomolecular complexes by TRF spectroscopy. Droplet microfluidics is used to produce with minimal material consumption an out-of-equilibrium biomolecular complex by rapid mixing of two reagents within water-in-oil droplets. TRF detection (ps to ns time scale fluorescence kinetics) is used as a reporter of the conformational heterogeneity along the relaxation of the system (ms to seconds reaction kinetics) during its propagation inside the microfluidic channel. In contrast to conventional stopped-flow experiments, which monitor the time-averaged fluorescence intensity, that is a signal averaged over all conformations, this approach does reveal conformational heterogeneity along the relaxation process through the distribution of fluorescence lifetimes. A streak camera is used to measure fluorescence decay kinetics with a 10-ps time resolution, along the microfluidic channel in which droplets propagate (Figure).

« Out-of-Equilibrium Biomolecular Interactions Monitored with Picosecond Fluorescence in Microfluidic Droplets», Maillot, S., A. Carvalho, J.-P. Vola, C. Boudier, Y. Mely, S. Haacke, and J. Léonard, Lab Chip, 14, p.1767-1774, 2014.

Funding: ANR FemtoSTACK (2011-14), PICO2 (2015-18)

Collaborations with Y. Mély (LBP, Unistra)

Figure: The fluorescence of droplets circulating in the microfluidic chip is imaged along the photocathode (PC) wire of a streak camera. 2D datasets are acquired in which the dimension along the PC wire is the chemical relaxation time axis (propagation in the microfluidic chip over seconds) and the second dimension is the fluorescence decay kinetics, which encodes the structural information (= distribution of conformations). From Maillot, S. et al. Lab Chip, 14, p.1767-1774, 2014.