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In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America . 8/25/2020, Vol. 117 Issue 34, p20566-20575. 10p. - Publication Year :
- 2020
-
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. [ABSTRACT FROM AUTHOR]
- Subjects :
- *SPIN labels
*DEER
*PROTEIN stability
*PROTEIN structure
Subjects
Details
- Language :
- English
- ISSN :
- 00278424
- Volume :
- 117
- Issue :
- 34
- Database :
- Academic Search Index
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 145306948
- Full Text :
- https://doi.org/10.1073/pnas.2005779117