Comparison of lipidic carrier systems for integral membrane proteins – MsbA as case study

Abstract

Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead to protein instability and reduced activity. Recently, lipid nanodiscs (NDs) and saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems that keep membrane proteins in a native-like lipidic solution environment and are suitable for biophysical and structural studies. Here, we systematically compare nanodiscs and Salipros with respect to long-term stability as well as activity and stability of the incorporated membrane protein using the ABC transporter MsbA as model system. Our results show that both systems are suitable for activity measurements as well as structural studies in solution. Based on our results we suggest screening of different lipids with respect to activity and stability of the incorporated IMP before performing structural studies.

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

“…In SANS, the use of deuterated samples allows contrast-matching techniques (Dunne et al, 2017;Laux et al, 2008;Haertlein et al, 2016) to provide unique information on protein-protein (Vijayakrishnan et al, 2010), protein-nucleic acid (Cuypers, Trubitsyna et al, 2013) or protein-lipid (Breyton et al, 2013) interactions. Sophisticated technologies have also been developed to allow the production of stealthdeuterated nanodiscs (Maric et al, 2014(Maric et al, , 2015 for the study of membrane proteins (Josts et al, 2018;Nitsche et al, 2018;Kehlenbeck et al, 2019). In the case of NR, a wide range of research now routinely exploits the contrast enabled through the use of selective deuteration (Grage et al, 2011;Waldie et al, 2018Waldie et al, , 2019Waldie et al, , 2020.…”

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

“…The flexibility of the Salipro technology has been demonstrated by the reconstitution of a multitude of IMP sizes and shapes, ranging from small monomeric proteins, such as the 16.5-kDa bacterial outer membrane protein OmpX (Chien et al, 2017), to large protein oligomers, illustrated by the tetrameric peptide transporter PepT So2 with a total of 56 transmembrane helices (Frauenfeld et al, 2016). The Salipro nanoparticles have been shown to be compatible with a wide range of high-resolution structural, biophysical and biochemical methods (Frauenfeld et al, 2016;Chien et al, 2017;Lyons et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019). Moreover, Salipro nanoparticles can be functionalized by a diverse range of tags on the SapA scaffold protein, useful for downstream applications relevant to pharmaceutical research.…”

“…Moreover, Salipro nanoparticles can be functionalized by a diverse range of tags on the SapA scaffold protein, useful for downstream applications relevant to pharmaceutical research. In addition, IMPs embedded in Salipro nanoparticles have been shown to be functional and can undergo conformational changes, highlighting the flexibility of the SapA scaffold to preserve the conformational changes required for protein function (Chien et al, 2017;Flayhan et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019).…”

“…In both cases, the reconstituted protein is shown to form pure, stable and homogenous Salipro nanoparticles. Previously, detergent-purified IMPs reconstituted into Salipro nanoparticles were reported to remain structurally intact and functionally active (Chien et al, 2017;Flayhan et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019). We envision that the DirectMX methodology will enable studies on IMPs that remained so far inaccessible to other solubilization or reconstitution methods, in particular IMPs representing unstable therapeutic targets from the families of GPCRs, ion channels and transporters.…”

“…Salipro nanoparticles are highly homogeneous, displaying both increased thermostability and half-life. In addition, Salipro-reconstituted IMPs have been shown to be functional and structurally intact by a wide range of biochemical and biophysical methods such as cryogenic EM, nuclear magnetic resonance spectroscopy or enzymatic assays (Frauenfeld et al, 2016;Chien et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019).…”