Invited
Residue specific conformational change and dimerization of proteins in cells determined by DEER measurement

The dynamics, associations and structures of proteins in cells are not necessarily the same as in in vitro conditions because the intracellular environment differs in viscosity, redox state, composition, and local confinement specific and non-specific interaction with cell components. Herein we use electron-electron double resonance (DEER) to delineate variations in protein oligomerization and conformation in cell, cell lysate and solution , all under freezing conditions. The target protein was the first baculoviral IAP repeats (BIR) domain of X-chromosome linked inhibitor of apoptosis (XIAP), BIR1. Site-specific labeling of BIR1 at different ligation sites, optimized by NMR spectroscopy, with rigid and stable BrPSPy-DO3A-Gd and BrPSPy-DO3MA-Gd tags allowed exploring the flexibility and structural variations of BIR1 in cells by double electron-electron resonance (DEER) distance measurements with high-spatial resolution. The results showed that the dimeric state of BIR1 persists in cells but with a higher dissociation constant and the increase was attributed to crowding effect based on comparison with in-vitro samples in the presence of the crowding agent ficoll. Moreover, parts of flexible N-terminal segment presents reduced flexibility in cells, in contrast to the disorder observed in-vitro and cell lysate. These results suggest that native intracellular condition can affect both reaction rates and the structural conformations of flexible polypeptide in cells.