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 KD 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.

Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: CuII–CuII and Nitroxide–Nitroxide Cases

“… 1 – 9 Due to these characteristics, PDS is also an emerging technique for conformational studies of protein and nucleic acid complexes in cellulo . 10 – 16 However, physiological concentrations are often in the sub-μM regime. In combination with low numbers of cells within samples, the challenge is to achieve sufficient absolute sensitivity.…”

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

“…Spin-labelled recombinantly produced soluble proteins have been already introduced into cells at micromolar concentrations using electroporation methods and interspin distances could be successfully detected (see, e.g., Ref. [94][95][96]). Sterically shielded nitroxide labels were also developed that have an increased lifetime in the reducing cellular environment [97][98][99][100][101][102][103][104][105].…”

Cosolute modulation of protein oligomerization reactions in the homeostatic timescale

“…The multiple mechanisms that simultaneously contribute to the nonideal behavior induced by the crowding agents is a reflection of the inherent complexity of the biological systems. For example, recent in-cell experimental evidence has reported a priori unexpected effects on the catalytic activity ( 13 ) and oligomerization ( 14 ).…”