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Pendant-bearing glucose-neopentyl glycol (P-GNG) amphiphiles for membrane protein manipulation: Importance of detergent pendant chain for protein stabilization.

ABSTRACT: Glucoside detergents are successfully used for membrane protein crystallization mainly because of their ability to form small protein-detergent complexes. In a previous study, we introduced glucose neopentyl glycol (GNG) amphiphiles with a branched diglucoside structure that has facilitated high resolution crystallographic structure determination of several membrane proteins. Like other glucoside detergents, however, these GNGs were less successful than DDM in stabilizing membrane proteins, limiting their wide use in protein structural study. As a strategy to improve GNG efficacy for protein stabilization, we introduced two different alkyl chains (i.e., main and pendant chains) into the GNG scaffold while maintaining the branched diglucoside head group. Of these pendant-bearing GNGs (P-GNGs), three detergents (GNG-2,14, GNG-3,13 and GNG-3,14) were not only notably better than both DDM (a gold standard detergent) and the previously described GNGs at stabilizing all six membrane proteins tested here, but were also as efficient as DDM at membrane protein extraction. The results suggest that the C14 main chain of the P-GNGs is highly compatible with the hydrophobic widths of membrane proteins, while the C2/C3 pendant chain is effective at strengthening detergent hydrophobic interactions. Based on the marked effect on protein stability and solubility, these glucoside detergents hold significant potential for membrane protein structural study. Furthermore, the independent roles of the detergent two alkyl chains first introduced in this study have shed light on new amphiphile design for membrane protein study. STATEMENT OF SIGNIFICANCE: Detergent efficacy for protein stabilization tends to be protein-specific, thus it is challenging to find a detergent that is effective at stabilizing multiple membrane proteins. By incorporating a pendant chain into our previous GNG scaffold, we prepared pendant chain-bearing GNGs (P-GNGs) and identified three P-GNGs that were highly effective at stabilizing all membrane proteins tested here including two GPCRs. In addition, the new detergents were as efficient as DDM at extracting membrane proteins, enabling use of these detergents over the multiple steps of protein isolation. The key difference between the P-GNGs and other glucoside detergents, the presence of a pendant chain, is likely to be responsible for their markedly enhanced protein stabilization behavior.

SUBMITTER: Bae HE

PROVIDER: S-EPMC7366829 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. STATEMENT OF SIGNIFICANCE: This study introduces new amphiphiles for use as biochemical tools in membrane protein studies. We identified a few hydrophilic pendant-bearing amphiphiles such as M-GTM-O11 and M-GTM-O12 that show remarkable efficacy for membrane protein solubilization and stabilization compared to a gold standard DDM, the hydrophobic counterparts (E-GTMs) and a significantly optimized detergent LMNG. In addition, detergent results obtained in the current study reveals the effect of detergent pendant polarity on protein solubility and stability. Thus, the current study represents both significant chemical and conceptual advance. The detergent tools and design principle introduced here advance protein science and facilitate structure-based drug design and development.

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Pendant-bearing glucose-neopentyl glycol (P-GNG) amphiphiles for membrane protein manipulation: Importance of detergent pendant chain for protein stabilization.

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