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Biochim Biophys Acta Biomembr. 2017 Nov;1859(11):2181-2192. doi: 10.1016/j.bbamem.2017.08.007. Epub 2017 Aug 10.

Purification and characterization of the colicin A immunity protein in detergent micelles.

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Instituto Biofisika (CSIC, UPV/EHU), Parque Científico de la UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain.
Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia Ed. 800, 48160 Derio, Spain.
Department of Biochemistry and Biophysics, Center for Biomembrane Research, The Arrhenius Laboratory, Stockholm University, 10691 Stockholm, Sweden.
Instituto Biofisika (CSIC, UPV/EHU), Parque Científico de la UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain; Departamento de Bioquímica, Universidad del País Vasco, 48940 Leioa. Spain.
Instituto Biofisika (CSIC, UPV/EHU), Parque Científico de la UPV/EHU, Barrio Sarriena s/n, 48940 Leioa, Bizkaia, Spain. Electronic address:


The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.


Circular dichroism; Colicin A immunity protein; Detergent; Membrane protein; Nuclear magnetic resonance; Protein purification; Size exclusion chromatography

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