Use of reverse micelles in membrane protein structural biology

Abstract

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques.

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

“…Integral membrane proteins have also been encapsulated and examined using high resolution NMR [23, 24, 58]. In a somewhat demanding case, the reverse micelle provided sufficient structural support to encapsulate the 54 kDa potassium channel KcsA in its functional form [23].…”

Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

“…3C). The use of reversed micelles was recently extended to a model membrane protein, gramicidin A, by Flynn and coworkers [167]. In their work dioctylsulfosuccinate (AOT)-based reverse micelles were solubilized in liquid pentane.…”

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

“…In recent years, reverse micelle encapsulation has been employed as a multidimensional NMR-based tool to characterize integral membrane proteins (Kielec et al 2009; Van Horn et al 2008), peripheral anchored membrane proteins (Valentine et al 2010) and proteins of marginal stability (Peterson et al 2004). It has also found application in protein and nucleic acid biophysics in a variety of contexts (Babu et al 2004; Nucci et al 2011; Pometun et al 2006; Simorellis and Flynn 2006; Van Horn et al 2005; Workman and Flynn 2009).…”