Peptergents:  Peptide Detergents That Improve Stability and Functionality of a Membrane Protein, Glycerol-3-phosphate Dehydrogenase

Abstract

Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl--D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.

Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

“…GlpD was expressed, detergent extracted, purified, and assayed as published (40). As detailed in the earlier report, detergent screening to maintain the activity of GlpD was conducted; for the structural and biochemical studies reported here, 1-O-noctyl-␤-D-glucopyranoside (␤-OG; MW 292.4) was used as the detergent.…”

“…Activity of GlpD was quantitated by determining the phenazine methosulfate (PMS)-coupled reduction of 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT; ϭ 17 mM Ϫ1 ⅐cm Ϫ1 ); this reduction depends on glycerol-3-phosphate. The reaction is measured spectrophotometrically at 570 nm in the presence of 1% ␤-OG, as described (40). The ability of MD and HQNO to function as the electron-coupler in place of PMS was quantitated.…”

Construction and Expression of Single Recombinant Peptide Surfactant for EOR Application

“…Peptide surfactant must have the characteristic that similar to surfactant which can be used in crystalization, that is peptide surfactant must be designed to have hydrophylic residual and 3 -6 hydrophobic residual at the tail (Yeh et al 2005 peptide is designed to have 3 amino acid residual of hydrophobic, that is arginine (R), lysine (K) and aspargarine (N). Prediction of protein structure of 1x SUPEL peptide that fused with Thioredoxine and TEV is analyzed in silico with bioinformatics program.…”

Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin

“…These peptide surfactants represent a promising approach to membrane protein crystallization. V 6 D was used to solubilize the integral membrane protein glycerol 3-phosphate dehydrogenase in solution, when assayed through protein gel electrophoresis, retaining its functional enzymatic activities (38).…”