Peptergents: Peptide Detergents That Improve Stability and Functionality of a Membrane Protein, Glycerol-3-phosphate Dehydrogenase † Joanne I. Yeh,* ,‡,§ Shoucheng Du, ‡ Antoni Tortajada, ‡ Joao Paulo, ‡ and Shuguang Zhang | Department of Molecular and Cell Biology, Brown UniVersity, ProVidence, Rhode Island 02912, Department of Chemistry, Brown UniVersity, ProVidence, Rhode Island 02912, and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ReceiVed July 13, 2005; ReVised Manuscript ReceiVed October 20, 2005 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-glucpyra- noside 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. Structural and functional studies of membrane proteins are often complicated by difficulties in obtaining active recon- stituted material (1, 2). Typical solubilization and purification procedures involve extracting the protein from the native membranes and reconstituting them into detergent micelles. Detergents, consisting of short-chain fatty acid amphiphiles, are routinely used in solubilization of proteins from native membranes (3). For crystal structure studies, detergents that form type II micellar structures, characterized by high critical micellar concentration (“CMC”), have been used most successfully for crystallization, although these detergents can have an adverse effect on protein stability. It is thought that the small micelle size allows these protein-embedded micellar surfaces to approach each other so that polar domains of proteins can contact, allowing for enhanced interaction to promote lattice formation (4, 5). Short peptides possessing detergent properties (“pepter- gents”) have been developed to self-assemble into well- ordered nanostructures and mimic some of the properties of lipid surfactant molecules (6). Polar interactions among headgroups are known to be necessary in the stabilization of the crystal lattice (7). As these peptergents possess optimizable chemical properties, mixtures of cationic and anionic peptides can result in unusual charge and polarity characteristics, leading to alteration of interactions between the peptides and proteins. Furthermore, peptergents form higher ordered structures in a concentration-dependent man- ner (8, 9). Peptergents typically consist of a short hydrophobic tail produced by repeat copies of nonpolar amino acids and a hydrophilic headgroup. The head can be either positively charged or negatively charged so that peptergents are consequently categorized as cationic or anionic detergents, respectively. Negatively charged heads consist of one or two aspartate or glutamate residues, and positively charged heads consist of one or two lysine, arginine, or histidine residues. In this study, V 6 D, V 6 K, A 6 D, A 6 K, and mixtures of A 6 D: A 6 K and A 6 D:V 6 K were used to solubilize and stabilize the enzymes of interest. V 6 D (VVVVVVD) is a peptergent that has biochemical properties that resemble those of lipids and other organic detergents. Structurally, in addition to its hydrophilic head and hydrophobic tail, V 6 D resembles a phospholipid mol- ecule in its length of approximately 2 nm (10). Functionally, V 6 D, along with other peptergents, resembles lipids in their † This project was funded from NIH (GM/AI66466, J.I.Y.), AFSOR/ MURI F49620-03-1-0365, J.I.Y./S.Z.), and March of Dimes Basil O’Conner Grant 5-FY00-564 (J.I.Y.). * Corresponding author. Current address: University of Pittsburgh Medical School; Department of Structural Biology; 3501 5th Avenue; Pittsburgh, PA 15260. Tel: (412) 648-9027. Fax: (412) 648-9009. Email: jiyeh@pitt.edu. ‡ Department of Molecular and Cell Biology, Brown University. § Department of Chemistry, Brown University. | Massachusetts Institute of Technology. 16912 Biochemistry 2005, 44, 16912-16919 10.1021/bi051357o CCC: $30.25 © 2005 American Chemical Society Published on Web 11/30/2005