Nouvelles stratégies de marquage paramagnétique pour l’étude des transitions structurales dans les protéines désordonnées – SPINFOLD

Protein dynamics and the structural changes they undergo are of fundamental interest since they are involved in a growing number of essential biological processes. This is peculiarly crucial for intrinsically
Disordered Proteins which play a wide variety of role, but which cannot be addressed by classical structural techniques. In this multidisciplinary
project gathering Chemists, Biologists and Physicists, four groups have joined their complementary expertise to design and develop new spin labeling probes for the EPR study of flexible proteins. Three proteins characterized by different levels of disorder have been choose as models: i) the NTail domain of the nucleoprotein of the measles virus; ii) the CP12 protein involved in CO2 assimilation by microalgae; iii) NarJ, a chaperone involved in the biogenesis of complex metalloenzymes. The synthesis of new nitroxide radicals able to be grafted in various protein sites, coupled to advanced EPR techniques provide essential structural information at nanoscale to understand conformation transition processes in
these systems.

Key results of the project are:
1/ the first grafting of a spin label on an amino acid other than a cysteine one: a tyrosine. An isoindoline based nitroxyde has been newly ynthesized,
selectively grafted on the tyrosine of a model protein and its ability to report structural modifications has been demonstrated.
2/ the synthesis and characterization of a phosphorylated nitroxide, allowing the diversification of EPR spectral signatures and consequently making possible the study of two sites simultaneously.
These results are a significant advance for the development of biostructural applications of EPR. The new spin labels synthesized in the project enabled significant progress in the understanding of molecular mechanism involved in recognition and induced fold processes in disordered proteins.
The most remarkable results are :
- Deciphering the induced folding of the NTAIL domain of the nucleoprotein upon interaction with the XD partner domain of polymerase in Measles virus and other closely related pathogen viruses.
- Evidence for a “fuzzy” complex involved in the regulation of the GAPDH enzyme by the small CP12 protein in the Calvin cycle for CO2 assimilation in milcroalgae. In this complex, although the C-term part of CP12 participates in the interaction between the two proteins, it keep a very high mobility at the electrostatically charged surface of GAPDH.
- Evidence fr a conformation selection mechanism in the interaction between the NarJ chaperone and its binding site in the final step of the biogenesis of the membrane bound molybdoenzyme nitrate reductase.

- optimize the grafting of spin labels targeting Tyrosine.
- Development of new spin labels with longelectronic relaxation times to enable long range distance measurements (> 6 nm) between magnetic probes by Double Electron Electron Resonance (DEER)
- Development of spin labels resistant to bioreduction to develop in cellulo EPR studies.
- Development of EPR methods to measure interspin distances between metal center and nitroxyde probes in metalloproteins. All these progress lead EPR spectroscopy to become a powerful technique to investigate
the structural and dynamic properties of large size or disordered protein systems, alternative of the classical structural approaches (NMR, X-ray crystallography).

The achievement of the project led to about 20 articles in international journals, one book chapter, and about 50 communication s in congress. Most of them associates 2 or 3 partners of the consortium, demonstrating a real team work in which sharing the skills of our multidisciplinary domains has been crucial for the success of the project. The novel spin
labels and the methodological progress lead us as leader in the field of biostructural EPR and several new collaborations have been initiated.

Monitoring structural transitions in proteins is of broad general interest because they are involved in many processes, such as enzymatic activity regulation, molecular recognition and assembly, and they are at the origin of multifunctionality properties. Such structural transitions are peculiarly important in Intrinsically Disordered Proteins (IDPs) which fulfil essential biological functions while lacking highly populated and uniform secondary and tertiary structure under physiological conditions.. The functional relevance of disorder resides in an increased plasticity that allows binding of multiple partners. Many IDPs adopt a well-defined conformation upon interacting with a target molecule, this disorder-to-order transition, referred to as 'induced folding', being tightly related to protein function. Thus, monitoring induced folding processes is essential to understand the functional roles of unstructured regions. However, due to the high flexibility of IDPs, induced folding is poorly accessible to investigations by conventional structural biology techniques, such as X-ray crystallography and NMR. Crystal-structure analysis cannot provide information on unstructured states, and NMR analysis is strongly impeded by the motional narrowing of the resonance dispersion in flexible systems. Other spectroscopic methods like Circular Dichroïsm (CD) and Small Angle X-ray Scattering (SAXS) can be used, but they only give global information on protein structure. Site Directed Spin Labeling (SDSL) followed by Electron Paramagnetic Resonance (EPR) is a particularly well-suited technique to study these transitions in highly flexible proteins. SDSL-EPR is based on the insertion of a paramagnetic label at a selected site of a protein, either on a native cysteine residue or on a cysteine introduced by site-directed mutagenesis, and the analysis of its mobility through EPR spectroscopy. It has the advantage of being not limited by the protein size and need weak amount of protein. Usual labels are nitroxide reagents and SDSL-EPR has been proved to be a useful technique to gain structural and dynamic information on protein-protein and protein-membrane interactions. However, the poor diversity of EPR spectral signatures given by commercially available labels and the frequent involvement of cysteine residues in biological functions are serious limitations of this approach. To date, only few attempts were made to extend the reporting capabilities of radical bearing molecular probe, and they remained dependent upon binding on cysteine. The aim of this project is to develop new paramagnetic labels allowing the diversification of their grafting sites as well as of their spectroscopic signatures in order to monitor conformational transitions in flexible systems. To reach this goal, four teams are combining their complementary skills and expertise in a highly multidisciplinary collaborative project, gathering chemists, biochemists, molecular biologists and physicists. In particular, new labels with specific isotopic coding of their EPR signatures (through magnetic nuclei such as 31P or 15N) will be synthesized, and new strategies to link covalently these novel spin labels to residues other than cysteines will be developed. This will allow the grafting of different labels either on the same protein or on two partner proteins and thanks to their different spectral signatures, to probe the local environment of each label independently. The conformational transitions will be monitored by EPR through the determination of parameters such as label mobility, solvent accessibility, and by inter-label distance measurements by pulsed EPR. As IDPs have an amino-acid compositional bias with a high proportion of polar and charged residues (Gln, Ser, Pro, Lys,'), and a low content of bulky hydrophobic amino-acids, this project focus specifically on Tyrosine and Tryptophan residues as new labeling targets. The scarcity of Tyr and Trp in IDPs is particularly well adapted to perform site directed labelling, and several strategies of spin label synthesis and grafting on these residues will be developed to optimize both the selectivity and yield of the spin labelling. Moreover, the larger temperature and pH stability range of IDPs, will enable to screen a high diversity of experimental conditions in order to optimize the grafting. The new spin labels will be used for studying the structural transitions of three proteins that exhibit various degrees of flexibility, and that are able to interact with different partners thereby modulating their properties: i) an intrinsically disordered protein domain (the NTAIL domain of the measles virus nucleoprotein); ii) a partly disordered protein (CP12, a protein involved in CO2 assimilation); iii) a folded protein harbouring several flexible regions (NarJ, a molecular chaperone, involved in the biogenesis of a metalloenzyme complex, namely the membrane-bound respiratory nitrate reductase).