DEER measurements of calmodulin : novel spin labels and structural insights

Double electron-electron resonance (DEER) spectroscopy is a biophysical technique used in structural biology. It allows nanometre scale distance measurements between spin labels containing unpaired electrons. In this thesis, DEER was employed to investigate the structure and behaviour of calmodulin, a calcium binding protein with significant functional and physiological importance. Following an introduction to the theoretical background and methodologies, the experimental work is organised around three main research questions. The first experimental chapter explores the use of bifunctional spin labelling of vicinal cysteines with a next-generation maleimide spin label. This approach aimed to reduce linker flexibility and to establish a viable labelling strategy for cysteine rich proteins. The second experimental section investigates the DEER characteristics of five Gd(III) spin labels, both well established and new molecules, measured on two calmodulin mutants, engineered to yield a short and respectively a long distance distribution. This study also compares sensitivity between two home-built W-band spectrometers and a commercial Q-band instrument. The final part examines the behaviour of calmodulin in a synthetic macromolecular crowding environment, combining DEER measurements of four apo and holo calmodulin mutants with computational modelling to assess the structural changes.