In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy.

ABSTRACT: The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd³⁺ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant K_D was markedly higher in cells than in solution and di

SUBMITTER: Yang Y 

PROVIDER: S-EPMC7456071 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature