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

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 Abbreviations: CD, circular dichroism; CFTR, cystic fibrosis transmembrane conductance regulator; DSC, differential scanning calorimetry; ESD, extramembranous soluble domain; IMAC, immobilized metal-affinity chromatography; IMP, integral membrane protein; NBD1, first nucleotide binding domain; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; SLS, static light scattering; TM, transmembrane. Detergent abbreviations (i.e. short names) are listed in Table I. Additional Supporting Information may be found in the online version of this article. 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.

Xkr8 phospholipid scrambling complex in apoptotic phosphatidylserine exposure

“…To assess molecular characteristics of the Xkr8 protein, PLB985 cells (PLB) not expressing Xkr8 (14) were transformed with Flagtagged human Xkr8 (hXkr8). Because the stability and subunit structure of membrane proteins is often regulated by Ca 2+ and detergent (19,20), PLB-hXkr8 was lysed in different detergents (CL47 or CL48) with mild and intermediate stringency (21) containing 0.5 mM EGTA or 1.0 mM Ca 2+ and separated by BN-PAGE. Western blot with anti-Flag showed that hXkr8 lysed in CL47 behaved as a large complex in the presence or absence of Ca 2+ (Fig.…”

A survey of detergents for the purification of stable, active human cystic fibrosis transmembrane conductance regulator (CFTR)

“…The shorter chain detergents have higher CMC values, and observed differences in ATPase activity might be due to increased detergent exposure at the necessarily higher detergent concentrations used during chromatography. Purification of CFTR in lysoPC, despite its low CMC, resulted in quite low reconstituted ATPase activity, suggesting loss of conformation such as that demonstrated when isolated NBDs of CFTR were exposed to this detergent [14]. Among widely used detergents, MNGs, DDM, and Chaps preserved the highest CFTR ATPase activities.…”

“…Another unique feature of CFTR is the R region whose phosphorylation regulates channel gating through an unknown mechanism [13]. CFTR NBDs exhibit conformational sensitivity to many detergents, likely contributing to the great difficulty in recovering active purified protein [14]. Purification of active CFTR from insect and yeast cells using perfluorooctanoate [15] or lysolipid [16–18] has been reported, but CFTR purified with these detergents in other laboratories has proven too unstable for detailed biophysical characterization.…”

Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies

“…A study of the CFTR-NBD1 indicated that ionic detergents could destabilize this domain [40]. Using NBD1 as a surrogate for full-length CFTR, the authors used differential scanning calorimetry (DSC) to investigate the effect of different detergents on the unfolding temperature of the purified protein.…”