Optimization of the design and preparation of nanoscale phospholipid bilayers for its application to solution NMR

Abstract

Despite arduous efforts and recent technological developments structural investigation of integral membrane proteins remains a challenge. The primary deterrents include difficulties with their expression, low inherent solubility and various problems associated with existing membrane mimicking systems. A relatively new class of membrane mimetics, nanodiscs, has been developed as a promising alternative. Although nanodiscs have been proven successful for several biophysical applications, they yet remain to become the system of preferred choice for structure determination. We have hereby made nanodiscs more suitable for solution NMR applications by reducing the diameter of the self-assembly complex to its potential limit. We achieved a noticeable improvement in the quality of NMR spectra obtained for the transmembrane and cytoplasmic domains of integrin αIIb incorporated into these smaller discs rendering them susceptible for a thorough structural investigation. We also present an on-column method for a rapid, efficient, single step preparation of protein incorporated nanodiscs at high concentrations. These discs have been fully characterized by transmission electron microscopy, dynamic light scattering and differential scanning calorimetry.

Advances in the use of nanoscale bilayers to study membrane protein structure and function

“…The truncation of MSP1D1 by selective removal of internal helices allowed for the reconstitution of bR and the β-barrel OmpX into nanodiscs as small as 6.3 nm in diameter, making them accessible to multidimensional NMR analysis and higher resolution structure determination (Hagn, Etzkorn, Raschle, & Wagner, 2013). In a related study, C-terminal truncations of MSP down to seven helical repeats rendered improved 15 N TROSY-HSQC spectral quality of integrin a IIB incorporated into nanodiscs, particularly for transmembrane residues (Puthenveetil & Vinogradova, 2013). Interestingly, both studies identified lower size limits for nanodiscs: below a certain MSP length threshold, discs lacked stability or began to form heterogeneous samples containing larger particles.…”

Chromatographic and electrophoretic methods for nanodisc purification and analysis

“…A final example for the use of affinity purification in the nanodisc preparation is its combination with nanodisc reconstitution in what is termed on-column reconstitution (Katayama et al 2010, Puthenveetil andVinogradova 2013). Here, either the MSP or the target membrane protein is immobilized on an affinity resin, after which the remaining of the reconstitution components such as lipids and detergents are added.…”

“…This approach allows for a single-step nanodisc reconstitution and purification. Despite the uncertainties in matching optimal reconstitution conditions such as MSP concentration on the column, recent reports have shown it to be feasible for the preparation of monodisperse nanodisc samples (Puthenveetil and Vinogradova 2013).…”

Nanodiscs and solution NMR: preparation, application and challenges

“…Preparing nanodiscs samples for NMR applications are time intensive and requires multiple purifications to separate empty discs from the incorporated ones. We developed a quicker, alternative, on-column method where the protein incorporated discs were formed on the resin either through the bound MSP (approach-1) or the bound target protein (approach-2) [19]. The advantages to this method were several-fold, including reduced preparation time, concomitant separation of empty and incorporated discs and better yields, which collectively improves the quality of our NMR spectrum.…”

“…Bacteriorhodopsin, a hepta-helical transmembrane protein, was studied comparatively across different mimetics comprising of micelles, amphipols, and (MSP1D1) nanodiscs [43]. 1 H- 15 N TROSY HSQC spectra of the trans-membrane and cytoplasmic domains of Integrin α IIb spectra was obtained in smaller D7 nanodiscs[19]. YgaP, an E. coli integral membrane protein composed of two transmembrane helices and cytoplasmic Rhodanese domain, was studied in mixed micelles, FC12 micelles, and MSP1D1 nanodiscs.…”