Towards Ligand-Induced Fluorescent Proteins – InducedFluo

The main properties will be obtained through a bottom-up approach going from the simulation of isolated ligand to the embedded ligand, with a strategy of increasing complexity through the four-year project. Of course, we will apply state-of-the-art multi-scale computational approaches. Several computational tools will be employed in order to fulfill the three objectives. More precisely molecular mechanics (MM) tools will be associated to both quantum mechanics (QM) methods and hybrid QM/MM methods. The dynamics of apoUnaG and holoUnaG proteins as well as the abilities of the BR to bind it will be evaluated through molecular dynamic simulations (MD) and free energy computations so to rationalize one of the most intriguing features of UnaG. In addition an extension of the non-empirical Effective Fragment Potential (EFP) approach will be considered for the first time onto a protein to account for the electronic polarization of the surrounding once the ligand is irradiated.

Long MD simulation of apoUnaG, holoUnag [wild-type (WT) and N57Q and N57A mutants] revaeled relevant features that can be related to explain the fluorescence spectra (its quantum yields). Indeed, in WT the ligand is stacked within Unag with stable interactions with two helices (H2 and H3) capping the beta-sheet barrel while a loop (L3) is slightly moving away. Within the barrel, bilirubin is making strong and stable interactions with T61, R112, Y134 along the simulations as well with water molecules. Such interactions lead to an almost rigid BLR geometry. In N57A and N57Q, the opposite is occurring, BLR is forming a novel interactions with Ser80 which is in L3 inducing an opening of the barrel (H2 and H3 slightly going away). The H-bond network around BLR is broken and therefore one of the pyrrole ring has much more free space inducing its rotations. Such flexibility in chromophore is known to reduce the quantum yield.

In addition, we have set up an application programming interface (API) named DesignFP with the automation of several redundant steps going from phylogeny, 3D model building, MD simulations, and docking a list of compound. The API is at the moment only available for our team team through github

Jan 2021 --> July 2021
Testing and benchmarking EFP onto the WT Xray structure in order to evaluated binging energies at EFP level of theory and absorption spectra at QM/EFP level of theory.
Aug 2021 --> Dec 2021
We will explore the anti-oxidant activities of BLR and several derivatives that could be docked into apoUnaG. In addition, we aim to improve (TD)-DFT using wavefunction approaches such as (SCS-)CC2. Deliverable: 1 article.
Enlarging QM/EFP calculations to simulations and to mutants.
Jan 2022 --> Dec 2022
Design of new ligand surroundings and docking of open-chain tetrapyrrole compounds. Deliverable: 1 article.
Binding free energy computations. Steered MD simulations have been already carried but are not optimal for such systems.

Conferences:

1. M. Asad, A. Laurent, Poster presentation, JTMS2020,
Exploring Structural Modifications of UnaG Fluorescent Protein Dynamics upon Mutations
2. A. Laurent, Invited Talk, GdR Photo Electro Stimulation Meeting (2020), Photochemical properties of molecular probes using quantum chemistry
3. A. Laurent, HDR Defense (2019), Modelling molecular and complexed surroundings properties

General conference:
A. Laurent, Obtention d’une ANR JCJC : témoignage, dans le cadre de l’Académie PULSAR (2020).

A new extremely promising class of ligand-induced fluorescent protein (LIFP) has been discovered very recently (June 2013). The UnaG protein emits intense and bright green light once the bilirubin (BR) ligand is bound to the protein. BR is an indirect marker of various human liver functions and has an antioxidant role. UnaG is therefore a promising direct medical tool to track liver disease as well as a probe to detect cardiovascular diseases. The InducedFluo proposal aims to model this new LIFP class for the very first time, to understand its, electronic, spectral and conformational properties as well as to design new ligands or mutants able to enlarge the panel of possible applications.

The InducedFluo goals will be fulfil thanks to the state-of-the-art multi-scaling computational tools including: i) robust and/or refined quantum mechanics methods to model the BR antioxidant and excited-states properties; ii) hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) schemes to account for surroundings; iii) molecular dynamics to assess the impact of conformational changes; iv) free energies calculations to estimate the binding energy of BR to the protein; and v) docking to explore new ligands, and so enhanced UnaG applications.
More precisely within the QM/MM methods we will use a very recent method to explicitly account for electronic polarization of the surrounding of the ligand. After a careful exploration of UnaG properties, we will suggest modifications of crucial neighbouring residues around BR to tune its response in order to enlarge the spectral activity of the BR once bound to the protein, e.g., going to the red will induce less cell damage. Eventually we should be able to associate UnaG to other human fatty acids compounds similar to BR that do not target the liver but other human functions.