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Protein Sci. 2014 Jun;23(6):769-89. doi: 10.1002/pro.2460. Epub 2014 May 3.

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

Author information

1
Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama; Center for Structural Biology, University of Alabama at Birmingham, Birmingham, Alabama.

Abstract

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.

KEYWORDS:

CD; CFTR; DSC; NBD1; detergent interaction; extramembrane domain; membrane protein; soluble domain; thermal unfolding

PMID:
24652590
PMCID:
PMC4093953
DOI:
10.1002/pro.2460
[Indexed for MEDLINE]
Free PMC Article
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