CE30 - Physique de la matière condensée et de la matière diluée

Mid-infrared femtosecond 2D spectroscopy in flavoenzymes by intrapulse difference frequency generation at 125 kHz – MIRTHYX

A new mid-infrared laser to help design tomorrow’s antibiotics

In order to reveal the flexibility and inhibition mechanisms of the bacterial enzyme ThyX, the MIRThyX project combines molecular biology methods, infrared laser technologies and femtosecond spectroscopy.

Unraveling the inhibition mechanisms of enzyme ThyX

Enzyme ThyX is a bacterial alternative to canonical Thymidylate Synthase ThyA, whose role is also to produce the nucleotide associated with the letter T in DNA. This enzyme is present in many pathogenic bacteria, but is absent in humans, making it a promising target for the development of a new generation of antibiotics. Indeed, inhibition of ThyX would mechanically inhibit the growth of the targeted pathogenic bacteria. Studies conducted over the last twenty years at the Laboratory of Optics and Biosciences (LOB) have produced very encouraging results allowing the identification of a certain number of molecules susceptible to inhibit ThyX. However, it now becomes necessary to better understand the underlying mechanisms at work, in particular by studying the effect of these potential inhibitors on the fluctuations of the protein structure. This is the objective of the MIRThyX project, conducted by LOB, Laboratoire Charles Fabry (LCF) and the company Amplitude Laser, in a pluridisciplinary framework involving laser technologies, femtosecond spectroscopy, and biotechnologies.

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 that can be addressed in the infrared, their association with 2DIR spectroscopy can bring combined spatial and temporal resolution to the investigation of protein conformational dynamics. Finally, new diode-pumped sources such as those developed by Amplitude Laser are revolutionizing femtosecond technologies, especially in combination with nonlinear optical techniques such as pulse compression and difference frequency generation. The MIRThyX project is based on these three pillars, the high rate and the stability of the infrared sources thus developed allowing the application of 2DIR spectroscopy to the study of ThyX, thanks to the use of local vibrational probes which will be substituted at strategically located amino acid residues of the ThyX protein.

The work carried out so far has focused on the optimization of the mid-infrared pulse source, with the design and test of various original generation methods. A new 2DIR spectrometer is also being developed in order to implement a fast delay scanning suitable for operation with high-repetition-rate diode-pumped lasers.

Beyond the study of the ThyX enzyme, which is the main objective of the project, the associated experimental development will make possible similar studies in many biochemical systems, e.g. for understanding enzyme active site dynamics. In addition, techniques of generation and characterization of infrared pulses as well as the new spectroscopies thus implemented could result in the development of innovative instruments.

No scientific articles have been published at this early stage of the project.

Due to its sensitivity to ultrafast fluctuations and to couplings between vibrational modes, two-dimensional infrared (2DIR) spectroscopy has provided during 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.

Project coordination

Manuel Joffre (Laboratoire d'optique et biosciences)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

AS AMPLITUDE SYSTEMES
LCF Laboratoire Charles Fabry
LOB Laboratoire d'optique et biosciences

Help of the ANR 460,707 euros
Beginning and duration of the scientific project: September 2019 - 48 Months

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