A genetically encoded ASAXS ruler to study the dimension of intrinsically disordered proteins

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Intrinsically disordered proteins (IDPs) lack persistent secondary or quaternary structures in their native states unlike folded proteins. Due to their dynamics, IDPs populate ensembles of interconverting structures. An important characteristic of such an ensemble is its scaling behavior describing how do the dimensions of the ensemble scale with the number of residues. From the perspective of polymer theory scaling contains the gist of preferential molecular interactions operative in a given ensemble; that is the scaling exponent suggests whether an ensemble is dominated by self-self-interactions or self-solvent interactions. From a biological standpoint, the scaling behavior largely enables one to predict biological and cellular functions of the proteins. For example, the recent years have seen an explosion of studies of liquid-liquid phase separation of IDPs or proteins bearing large disordered segments. Such phase behavior can often be explained through the formalisms of phase separation in polymers, and the dimensions and scaling of the IDP ensembles play a crucial role here. The most common experimental observables used to assess conformational aspects of the ensembles are RG (radius of gyration) typically obtained from small-angle X-ray scattering (SAXS) and RE (end to end distance) measured from fluorescence resonance energy transfer spectroscopy (FRET), especially single molecule, smFRET. The fact that the two methods disagree has been an ongoing debate, which eclipsed last year in a number of conflicting publications in high impact journals on the origin of this phenomenon. Notably, the debate challenges an entire field, not just single papers. We propose a residue specific anomalous SAXS (ASAXS) approach enabling to measure RG and RE from the same sample and provide the ultimate answer to this debate. We will develop novel genetic code expansion techniques to insert small anomalous X-ray scatters at exactly the same position into a large set of proteins, where in smFRET dye labels are placed. This will be paired with advanced tools development to extract the weak anomalous signal from single atom based scatters, and the ASAXS experiments will be conducted at the arguably world most advanced biological SAXS beamline. We will advance the new ASAXS-based method further beyond just resolving the IDP debate to demonstrate its ability to measure large distances in dynamic proteins beyond scales currently accessible by any technique, thus generating a new integrative structural biology tool.