Reconstitution of cholesterol-dependent vaginolysin into tethered phospholipid bilayers: implications for bioanalysis

PloS One
Rima BudvytyteGintaras Valincius

Abstract

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity in vitro and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the t...Continue Reading

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Citations

Jul 30, 2015·Langmuir : the ACS Journal of Surfaces and Colloids·Daniel R ScottHirsh Nanda
Dec 15, 2016·Chemistry and Physics of Lipids·A ValiūnienėG Valinčius
May 24, 2016·Biochimica Et Biophysica Acta·Giulio PretaGintaras Valincius
Apr 18, 2020·Biochimica Et Biophysica Acta. Biomembranes·Tadas PenkauskasGiulio Preta

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Methods Mentioned

BETA
dynamic light scattering
PCR
enzyme-linked immunoassay
ELISA
circular dichroism
Circular

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