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Pulsed Dipolar Spectroscopy on Symmetric Oligomers

Final Report Summary - OLIGO PELDOR (Pulsed Dipolar Spectroscopy on Symmetric Oligomers)

Biomolecular and biomedical research is driving towards dissecting macromolecular assemblies of ever increasing complexity. Understanding such complex structures and the structural changes involved during function promises great opportunities for new molecular approaches in health and disease. However, resolution of atomistic detail in large molecular complexes and assemblies is not always feasible for all functional conformations involved. Here, the emerging approach of pulse EPR distance measurements is of particular strength as it allows simplifying the complex structure under study using a number of selectively introduced probes and their interactions.
In this approach, a paramagnetic tag or label is attached to the protein or nucleic acid, and acts as a ‘beacon’ illuminating its immediate surroundings and allowing assessing the number of probes involved, as well as the distances between them.

Especially the pulsed electron-electron double resonance (PELDOR or DEER) method is becoming increasingly applied. The method is particularly promising for structural studies on different functional states of membrane proteins which are difficult to access by most biophysical methods, including X-ray crystallography, cryo electron microscopy and nuclear magnetic resonance spectroscopy. However, in samples of membrane proteins several approximations commonly made in data analysis might not be well met, which could impact interpretability and reliability of the results obtained. Especially the presence of more than two tags per macromolecular complex poses problems in analysis with implications for the design of novel experiments. In this project, a number of new chemical model systems that mimic multiply tagged biomolecules have been designed and synthesised. These have been used to analyse how the shortcomings of the current experimental designs and analysis approaches for the PELDOR experiment could be overcome. The results obtained on these chemical models are in the process of being transferred and applied to several proteins of interest, all of which are forming multimers (from dimers to octamers), to aid the understanding of their structure and function. Furthermore, a number of experimental approaches allowing to diminish problems introduced by the presence of multiple tags have been evaluated and benchmarked.

This project has provided the armoury of biophysical methods with insights that allow a more precise use of pulse EPR spectroscopy for distance measurements in multimeric proteins in solution and bound to membranes. All these considerations have been tested on well-defined model complexes synthesised within the framework of the project and have been transferred to cutting-edge structural investigations on biomedical targets also involving membrane proteins of unknown structure.