MIRThyX
Mid-infrared femtosecond 2D spectroscopy in flavoenzymes by intrapulse difference frequency generation at 125 kHz
Due to its sensitivity to ultrafast fluctuations and to couplings between vibrational modes, two-dimensional infrared (2DIR) spectroscopy has provided over the last twenty years a wealth of information on chemical and biochemical reactions. In parallel, recent progress in biotechnology now allow the insertion of unnatural amino acids at specific sites of proteins. As some of these unnatural amino acids can serve as local vibrational probes, their association with 2DIR spectroscopy can bring combined spatial and temporal resolution to the investigation of protein conformational dynamics. However, the development of this emerging approach has been hindered by signal-to-noise and data throughput limitations associated with current 2DIR spectrometers, most often based on 1-10 kHz Titanium:Sapphire femtosecond amplifiers. The aim of the MIRThyX project is to address this technical barrier by developing a new-generation 2DIR spectrometer that will take advantage of the recent advent of diode-pumped ultrashort lasers, and to apply this new instrument to the investigation of the structural dynamics of the flavoenzyme ThyX. The project will be carried out by three partners: two academic partners, Laboratoire d’Optique et Biosciences (LOB) and Laboratoire Charles Fabry (LCF) and one industrial partner, Amplitude Systèmes (AS). The remarkable complementarity of the partners will thus cover the broad range of skills needed for undertaking this interdisciplinary project, involving laser technology, mid-infrared (MIR) pulse generation and characterization, 2DIR spectroscopy and biochemistry.
A great asset of the MIRThyX project is a recent breakthrough, published in early 2019 by two of the partners (LCF and AS), consisting in the efficient temporal compression down to 7 fs of 300-fs pulses delivered by an Ytterbium Doped Fiber Amplifier. The spectral bandwidth of the high-energy pulses thus achieved is sufficiently broad to directly generate MIR pulses by intra-pulse Difference Frequency Generation. This approach will enable the generation of MIR pulses in a straightforward, single-beam arrangement at a repetition rate of 125 kHz, which is optimal for equilibrium 2DIR spectroscopy. This MIR femtosecond source will then be combined with a compact and fast-scanning interferometer associated with a Chirped-Pulse Up-conversion detection stage, resulting in excellent resolution in both pump and probe dimensions. The resulting 2DIR spectrometer, of unprecedented signal-to-noise ratio, spectral bandwidth and spectral resolution, will be demonstrated with the investigation of the structural dynamics of flavoenzyme ThyX, a bacterial alternative to Thymidylate Synthase, whose purpose is to produce the nucleotide associated with the letter T in DNA. Flavoenzyme ThyX being present in many pathogenic bacteria, while being absent in humans, it constitutes a promising antimicrobial target. In association with molecular dynamics simulations, multidimensional spectroscopy will help us understanding how some substrates or inhibitors act on structure fluctuations at different sites of interest determined by the chosen location of our vibrational probe. Our research might thus contribute to the optimization of new inhibitors.
Beyond its scientific impact on 2DIR spectroscopy in proteins, the MIRThyX project will also result in a series of instrumental developments in terms of MIR pulse generation, characterization and application to 2DIR spectroscopy. The participation of the industrial partner will thus be essential, not only in the development of the MIR source itself but also to facilitate possible commercial development of the innovative instruments that will be designed during the project.
At this stage, we have developed a 100-kHz tunable MIR source, as shown in the graph below.
Our work on MIR detection at 100 kHz and application to pump-probe spectroscopy has been presented at TRVS 2023.
Publications
Q. Bournet, F. Guichard, M. Natile, Y. Zaouter, M. Joffre, A. Bonvalet, I. Pupeza, C. Hofer, F. Druon, M. Hanna, P. Georges, Enhanced intrapulse difference frequency generation in the mid-infrared by a spectrally dependent polarization state, Opt. Lett. 47, 261 (2022).
Q. Bournet, M. Jonusas, A. Zheng, F. Guichard, M. Natile, Y. Zaouter, M. Joffre, A. Bonvalet, F. Druon, Marc Hanna, P. Georges, Inline amplification of mid-infrared intrapulse difference frequency generation, Opt. Lett. 47, 4885 (2022).
Q. Bournet; M. Natile; M. Jonusas; F. Guichard; Y. Zaouter; M. Joffre; A. Bonvalet; F. Druon; M. Hanna; P. Georges, Intensity noise in difference frequency generation-based tunable femtosecond MIR sources, Opt. Expr. 31, 12693 (2023).