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 dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability. Significance The question how does the intracellular environment affect the structure, dynamics, and stability of a protein and protein–protein interactions has been a center of interest in the context of understanding proteins function in their native environment. We addressed this question by exploring the conformational space and the stability of a homodimeric protein complex in human cells as compared to in dilute buffer solution and cell lysates. We showed that the dimer is destabilized in the cells, which is opposite to the theoretical predictions. Based on complementary in vitro measurements, we suggest that the destabilization originates from a combination of the crowding and shape of the dimer, the higher ionic strength in the cell, and potential attractive chemical interactions.

The reaction of phenylsulfonated pyridine derivatives and protein thiols is suitable for high-resolution spectroscopic analysis by generation of a rigid, stable and short thiolether tether.