Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study: Implications of Detergent Kink for Micellar Properties

Abstract

Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-β-d-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein-stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.

Solution Properties of Alkyl β‐D‐Maltosides

“…Therefore, they have a wide range of applications and development in food, cosmetic, pharmaceutical industries, and scientific research (de Foresta et al, ; Jastrzebska et al, ; Ni et al, ; Pillion et al, ; Raman et al, ; Rifkin et al, ; Santonicola et al, ; Weber and Benning, ; Zhang et al, ). They have been used in drug delivery (Ahmad et al, ), thermal stabilization (Ahmad et al, ), hydrolytic metabolism (Weber and Benning, ), salt tolerance (Zhang et al, ), formation of higher‐ordered structure (Jastrzebska et al, ), co‐immunoprecipitation (Lee et al, ), and structure analysis (Ni et al, ), as well as therapeutic adjuvants (Rifkin et al, ), permeation enhancers (Pillion et al, ), absorption enhancers (Gradauer et al, ), and membrane protein stabilizers against denaturation (Das et al, ; de Foresta et al, ; Jastrzebska et al, ; Klammt et al, ; Newby et al, ; Raman et al, ; Santonicola et al, ; Whorton and MacKinnon, ).…”

“…Weber and Benning, 1984;Zhang et al, 1996). They have been used in drug delivery (Ahmad et al, 2014), thermal stabilization (Ahmad et al, 2014), hydrolytic metabolism (Weber and Benning, 1984), salt tolerance (Zhang et al, 1996), formation of higher-ordered structure (Jastrzebska et al, 2006), co-immunoprecipitation (Lee et al, 2018), and structure analysis (Ni et al, 2011), as well as therapeutic adjuvants (Rifkin et al, 2011), permeation enhancers (Pillion et al, 2002), absorption enhancers (Gradauer et al, 2017), and membrane protein stabilizers against denaturation (Das et al, 2017;de Foresta et al, 2011;Jastrzebska et al, 2006;Klammt et al, 2012;Newby et al, 2009;Raman et al, 2006;Santonicola et al, 2008;Whorton and MacKinnon, 2013).…”

New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies

“…, acyloxonium ion), known as anchimeric assistance, all glycosidic bonds were expected to be β. 27 The stereochemistry of the glycosylated products could be readily confirmed by 1 H NMR spectra of the final amphiphiles dissolved in CD 3 OD. The proton attached to the anomeric carbon (C1) was coupled to the neighboring C2 proton with a vicinal coupling constant ( 3 J ) of 8.0 Hz as both protons are in the axial position.…”

“…As for β 2 AR, the receptor in MNG-3 retained ~60% ligand binding ability after a 5-day incubation at room temperature in a previous study. 27 Under the same conditions, the TPS-E10L-solubilized receptor retained >85% activity. In the case of MelB St , the transporter extracted by TPS-E7 was more responsive to galactoside binding than the protein obtained in MNG-3.…”

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

“…[24] Theaggregated material was then removed by centrifugation and the supernatant submitted to SDS-PAGEa nd Western blot analysis to quantify the remaining solubilized MPs.Afull experiment is displayed in Figure 2B and Dfor each protein fraction incubated with 8b, allowing the apparent melting temperatures, T m ,a tw hich 50 %ofBmrA or A 2A Rremains in solution (dotted lines), to be estimated. [9] This result positions DCODs at the forefront of the very small set of stabilizing detergents,s uch as LMNG, [15] FA3, [17] and the recently published norbornane-based maltosides (NBMs), [25] that are able to generate an increase in T m of more than one log unit. Aclear effect was observed with respect to the hydrophobic component, namely the larger,the better.Note that this gain in thermostability is equivalent to that obtained previously by introducing 17 mutations in am altose-binding protein-A 2A Rf usion protein with aC -terminal 96 residue truncation.…”