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Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

ABSTRACT: Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the protein's functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.

SUBMITTER: Etzkorn M

PROVIDER: S-EPMC3595385 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

Etzkorn Manuel M Raschle Thomas T Hagn Franz F Gelev Vladimir V Rice Amanda J AJ Walz Thomas T Wagner Gerhard G

Structure (London, England : 1993) 20130214 3

Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the hept ...[more]

PMID: 23415558

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Project description:Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the apolipoprotein B gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.

Dataset Information

Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

Publications

Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

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