Final Report Summary - ANISOPROTEINXRAY (A structural mechanism of photoreceptor protein signalling by time-resolved anisotropic X-ray solution scattering) The project ANISOPROTEINXPAY aimed to elucidate the structural changes that relay the response of a photoreceptor protein towards a cellular response by time-resolve X-scattering in solution. In order to further promote X-ray scattering in solution, the researcher proposed to improve the information content of these data by developing a novel sample environment that restrict over all protein motion, while allowing for large conformational changes of the protein.In the beginning of the project period, a publication of structural changes in the photoreceptor protein that was supposed to be investigated proved the structural change relatively small and therefore unsuitable for the proposed work [Winkler 2013 J.Mol.Biol.]. Therefore, the researcher took part in photoreceptor studies being done in the work group as a part of the experimental team needed for synchrotron and even XFEL-experiments (2 manuscripts in preparation). In this way she was trained as proposed, missing the overexpression and purification of a protein. The major forces were used towards the proposed method development (Objectives Method III and IV). Aiming to establish improved time-resolved X-ray scattering in solution, proteins were entrapped in hydrogels, and their mobility was tested using spectroscopy. For this, various methods to produce suitable hydrogels were tested, evaluated and improved. A major requirement was to be able to load sufficient amounts of proteins without affecting their function and possibility to undergo major structural changes upon illumination with laser light. In three synchrotron beamtimes (APS 2014, Maxlab 2014, and ESRF 2015) the protein loaded hydrogels were tested and data analysis was carried out to the point that manifested low signal to noise ratios. Main results and their impactThe main achievement within the first year of the project was to enable rotational restriction of functional photoreceptor proteins by two types of highly specific links to hydrogels. All proteins contained in the processed sample are held in 2 positions of their sequence.Also, major obstacles in time-resolve anisotropic X-ray scattering on proteins in solutions are identified and might lead to successful implementation of this method.In a similar project conducted more independently by the funded researcher in a collaboration with both Umea University Sweden and Halle University, Germany, the structural mechanism of am membrane remodelling protein could be established. Additionally, the role of ATP within the functional cycle of the protein could be deciphered. These findings explain major unknown steps in clathrin-independent endocytosis as it is related to membrane trafficking and receptor recycling (Manuscript in preparation).
