SPINFOLD

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).