期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 34, 页码 20566-20575出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2005779117
关键词
DEER; XIAP; in cell measurements; Gd(III) labeling
资金
- National Natural Science Foundation of China (NSFC)-Israel Science Foundation [21761142004, 2484/17]
- Major National Scientific Research Project in China [2016YFA0501202]
- NSFC [21673122, 21473095]
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.
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