The cytoskeletal protein vinculin plays a key role in the control of cell-cell or cell-matrix adhesions. It is involved in the assembly and disassembly of focal adhesions and affects their mechanical stability. While many facts highlight the importance and significance of vinculin for vital processes, its precise role in the regulation of cell adhesions is still only partially understood. Various EPR methods are used in this work in order to study the vinculin tail (Vt) domain in an aqueous buffer solution and its structural changes induced by F-actin and acidic phospholipids. EPR results in combination with a rotamer library approach (RLA), MD simulation and other computational methods allowed the construction of molecular models of Vt and dimeric Vt in the presence and absence of its binding partners. Furthermore, X-band orientation selective DEER measurements were applied on a Vt double mutant. It could be shown that the determination of the mutual orientation of protein bound spin labels is possible at X-band frequencies, if the orientation correlation of the spin label pair is strong. The method established here can be used to determine valuable information about proteins and nucleic acids, expanding the virtue of DEER spectroscopy as a tool for structure determination.

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