Detergent Systems Research Articles

Human Claudin-7 cis-Interactions Are Not Crucial for Membrane-Membrane (Trans-) Interactions.

Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. In vivo, these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= cis-interaction), and also interact with Cldns from adjacent cells (= trans-interaction). Interactions of Cldns are typically studied in vivo and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in E. coli and purification of human Cldn7, enabling in vitro analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 cis-interactions. Furthermore, we identified two amino acids which mediate electrostatic cis-interactions and analyzed the impact of disturbed cis-interaction on trans-contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 cis-oligomerization might not be a prerequisite for establishing trans-contacts.

In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase.

Glycoproteins are post-translationally modified proteins that take part in nearly every biological process and make up a large percent of the proteome. N-Linked glycosylation can be performed by N-glycosyltransferase (NGT), which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT), within the protein. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Therefore, the use of in vitro protein glycosylation can help further evaluate the effects of the properties for these important macromolecules. In vitro studies of N-linked glycosylation were done in a stepwise fashion in a membrane-mimetic environment to confirm that the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used since hydrophobic peptides and membrane proteins are insoluble in aqueous solvents. The stepwise method consisted of the glycosylation of a soluble 7-residue peptide, a hydrophobic WALP-NVT peptide, and a γ-sarcoglycan membrane protein, all of which contained the glycosylation site Asn-Val-Thr (NVT). Glycosylation of the samples was performed using Escherichia coli-expressed NGT from the Actinobacillus pleuropneumoniae genome, and a single sugar moiety of glucose, provided from a nucleotide-linked donor, was added to the glycosylation site. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection of glycosyltransferase activity and to show the attachment of a single glucose molecule. Our experiments demonstrated that small or large membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by NGT in membrane-mimetic environments.

Переосмысление парадигмы оценки качества кормов

The schema of a chemical analysis of the forages, developed in 1860 by Wilhelm Henneberg in the village Weende (Holland), is applied in our country till present time almost without changes. A basis of the forages evaluation test is the organoleptic, physical and chemical indexes of the crude nutrients content (dry matter, protein, fiber, fat, ash); plus for ensilage forages — рН, the content of organic acids, ammonia nitrogen; for artificially dried forages — the physical form, carotin etc. The last decade are spreaded also commercial laboratories from developed countries having century traditions of the production quality estimation. Instead of Weende model comes the detergent schema of Van Soest allowing to refuse crude ("unpurified") nutrients and to pass on to pure substrates of carbohydrates — unstructured (sugar, starch and so on), easy and difficult digestible (hemicellulose, cellulose) and to practically undigestible polymer lignine. In this article has be done comparative analysis of traditional Weende and detergent systems, using data of laboratory BLGG AgroXpertus and a concrete example of evaluation test of corn green mass and the silage prepared from it. Despite different approaches and amount of used indexes comparison has shown adequacy of domestic and foreign systems of these forages evaluation. However, the accumulated experience demands to be detailed assimilated, and the advanced methods should find worthy application in our laboratory practice.

Modeling detergent applications using molecular dynamics

The molecular dynamics group at Fairmont State University models detergent systems with interdisciplinary applications. Simulations currently underway include octane interacting with sodium dodecyl sulfate (SDS) in aqueous solution as well as micelle formation in systems of cetyltrimethylammonium bromide and chloride (CTAB and CTAC, respectively). The simulation of octane and SDS in aqueous solution is designed to model the process of washing away grease with a detergent commonly found in dish soap. It is intended for use as a web-based educational tool to provide a dynamic visualization of micelle formation, e.g. in freshman chemistry classes. CTAB was chosen as a target because of literature evidence that low concentrations of the detergent cause a 100-fold increase in fluorescence of a small organic molecule. This organic molecule is a localized photosensitizer, which is useful in improving photodynamic light therapy that is currently being used as treatment for some cancers. Bromide ion parameters were adapted from the well parameterized chloride ion in the CHARMM forcefield; the comparison is interesting because CTAC and CTAB are known to have different aggregation numbers. Initial simulation results indicate that micelle formation from a random assembly of detergent molecules (n=20-130) occurs rapidly for both species. This research has been supported by the Fairmont State University College of Science and Technology as well as the NASA WV Space Grant Consortium.

An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases.

Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.

Action of surfactants on the mammal epidermal skin barrier.

Daily skin washing routines can promote undesirable effects on skin barrier function. The stratum corneum (SC) lipid matrix is crucial for skin barrier function. Skin cleansing products are mostly composed of surfactants: surface-active molecules that interact with skin lipids in several ways. The main aim of this work was to investigate the effect produced by surfactants on skin barrier permeability. Porcine skin is a well-accepted and readily available model of the human skin barrier. The effect of two cleansing formulations (based on different surfactant mixtures) on the barrier properties of mammalian skin were evaluated. Water sorption/desorption (DVS) experiments were used to measure skin permeability. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and confocal Raman were useful to study SC lipid organization. The results showed that while anionic surfactants (SLS) had a negative impact on the skin barrier, with a clear increase of alkyl chain disorder; cosurfactants present in the shampoo formulation diminished the detrimental effect of their primary ionic surfactant, inducing less modification on lipid intramolecular chain disorder. The obtained results confirmed that the mild cleansing formulations studied had gentle interaction with skin. The capacity to discriminate between detergent systems was clearly established with both DVS and spectroscopy techniques.

Feasibility of Cryo-EM for Structural Characterization of Small Membrane Proteins

Single-particle cryo-EM has recently emerged as a tool for characterization of protein structure at atomic resolution, with applications both to soluble and membrane proteins. Cryo-EM relies on computational alignment of single-particle images, permitting calculation of interpretable two-dimensional averages and three-dimensional reconstructions. Prior to data collection, cryo-EM samples are trapped in vitreous ice by plunge freezing, protecting biological macromolecules from radiation damage and high-vacuum, yet maintaining hydration and native structure. Soluble proteins are thus surrounded by water, which has a small electron scattering cross-section relative to the protein itself. However, membrane proteins in detergent or lipid nanoparticle systems (nanodiscs, saposin), are embedded in contrastive and predominantly unstructured material. The impact of this contrastive, unstructured mass on image alignment and three-dimensional reconstruction is unknown. Indeed, traditional cryoprotectants (glycerol) are not used in cryo-EM because their protein-like scattering is deleterious to particle contrast. Nevertheless, a growing number of membrane proteins have been characterized using cryo-EM. To date all such proteins bear large, soluble domains (often dwarfing the membrane bound component, e.g. nanodisc-anchored ribosomes), which are hypothesized to drive image alignment. Recent structures of TRPV1 (a tetrameric Ca2+ channel) reconstituted in lipid nanodiscs show that current cryo-EM pipelines can deliver high resolutions even when the ratio of structured to unstructured mass falls as low as 2:1. Here, we undertake to ascertain the limits to this ratio (if any), as well as the feasibility of reconstructing structures lacking any soluble extension.Starting from micrographs of TRPV1 in lipid nanodiscs, we use signal subtraction to simulate data for progressively smaller ratios of structured to unstructured mass, eventually eliminating all contributions from the soluble domains. Our results, including a 3.4 Angstrom structure of a hypothetical membrane protein with no soluble domain, suggest that high-resolution alignment and reconstruction is feasible for membrane proteins with no soluble extension, including when the ratio of structured to unstructured mass is less than 1:1. With continued technological development, it may be possible to adopt cryoprotectants that greatly enhance the formation of vitreous ice even during slow freezing. Finally, this work also serves as a tutorial for precise signal subtraction using open-source software, including creation of a suitable subtraction map, simulation of particle image contributions, recentering of subtracted particles, and per-particle normalization based on Fourier correlations.

Concentrating membrane proteins using ultrafiltration without concentrating detergents

Membrane proteins (MPs) are of rapidly growing interest in the design of pharmaceutical products, novel sensors, and synthetic membranes. Ultrafiltration (UF) using commercially available centrifugal concentrators is typically employed for laboratory-scale concentration of low-yield MPs, but its use is accompanied by a concomitant increase in concentration of detergent micelles. We present a detailed analysis of the hydrodynamic processes that control detergent passage during ultrafiltration of MPs and propose methods to optimize detergent passage during protein concentration in larger-scale membrane processes. Experiments were conducted using nonionic detergents, octyl-β-D glucoside (OG), and decyl-β-D maltoside (DM) with the bacterial water channel protein, Aquaporin Z (AqpZ) and the light driven chloride pump, halorhodopsin (HR), respectively. The observed sieving coefficient (So ), a measure of detergent passage, was evaluated in both stirred cell and centrifugal systems. So for DM and OG increased with increasing filtrate flux and decreasing shear rates in the stirred cell, that is, with increasing concentration polarization (CP). Similar effects were observed during filtration of MP-detergent (MPD) micelles. However, lower transmission was observed in the centrifugal system for both detergent and MPD systems. This is attributed to free convection-induced shear and hence reduced CP along the membrane surface during centrifugal UF. Thus to concentrate MPs without retention of detergent, design of UF systems that promote CP is required. Biotechnol. Bioeng. 2016;113: 2122-2130. © 2016 Wiley Periodicals, Inc.

Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry.

Aspirin and other nonsteroidal anti-inflammatory drugs target the cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the monooxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for the addition of oxygen to the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9, 2.0, and 2.4 Å, respectively. The structures reveal that (1) the acetylated Ser-530 completely blocks access to the hydrophobic groove, (2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation, and (3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove. On the basis of these structural observations, along with functional analysis of the S530T/G533V double mutant, we propose a working hypothesis for the generation of 15R-HETE by aspirin-acetylated COX-2. We also observe differential acetylation of COX-2 purified in various detergent systems and nanodiscs, indicating that detergent and lipid binding within the membrane-binding domain of the enzyme alters the rate of the acetylation reaction in vitro.

Insights into the Stability of GPCRs in Detergent Micelles

G-protein-coupled receptors (GPCRs) are highly dynamic membrane proteins and therefore optimization of detergents for solubilization and purification of GPCRs is an arduous task for extraction from cell membranes. The structural dynamics of GPCRs in detergents will provide key insights into the thermodynamic basis of GPCR stability in detergents. Here we report the structural insights into the stability of adenosine receptor A2AR in various detergent systems with varying tail length. We performed microseconds of atomistic molecular dynamics simulations of the A2AR in the inactive and active-like conformational states, in detergent micelles of DDM (Dodecylmaltoside), DM (Decylmaltoside), NG (Nonylglucoside), and OG (Octylglucoside). The calculated stability of the A2AR in these four detergents correlates well with the experimentally measured stability. Further analysis showed that the A2AR in DDM is less flexible and less dynamic compared to the same receptor in short tail detergents such as NG, and OG micelles. The DDM detergent preserves the helicity of the transmembrane helices in A2AR compared to other short chain detergents. NG and OG systems with shorter alkyl tails lead to unraveling of protein structure and abnormal insertion of detergent into the protein that denatures the functional conformation. We find that longer hydrophobic detergent DDM simulation more closely resembles the bilayer simulations in terms of energetics and interaction dynamics, and the insights gained from this study can be used to optimize detergents prior to experiments for GPCRs solubilization.

Folding and Stability of Transmembrane b-Barrels of Bacterial and Human Origin: Probing Underlying Similarities and Principal Differences Using In vitro Systems

The biochemical, biophysical and functional characterization of transmembrane β-barrels of human and bacterial origin has provided insight on factors that stabilize these proteins in various lipid and detergent systems. This review attempts to summarize our recent findings and contributions to the field of membrane protein biophysics. Using various β-barrels, particularly, the 8-stranded OmpX and Ail from Escherichia coli and Yersinia pestis, respectively, and the 19-stranded human mitochondrial transmembrane barrel VDAC-2 (the voltage-dependent anion channel isoform 2) as model systems, we have observed that barrel-micelle interaction strengths, protein-to-detergent ratios and interface residues serve as key elements in determining the stability of these barrel systems, irrespective of the protein origin. Differences in the behaviour of these barrels arise from their malleability to reversible folding versus stabilization by kinetic contributions, with the latter giving rise to irreversible unfolding and aggregation. Furthermore, while bacterial proteins are more tolerant to mutations, hVDAC-2 is particularly influenced by subtle (conserved) mutations, both in its stability and function. The implications of these findings to the field of membrane proteins are also discussed.

Short Chain Lipids Maintain Adenosine A2AR Ligand Binding in the Absence of Cholesterol

Evidence of preferential interactions between sphingolipids and cholesterol, and the identification of cholesterol binding pockets in some membrane proteins highlights the importance of lipids in the regulation of membrane protein function. However, the mechanism through which these lipid-protein interactions modify the conformation and activity of membrane proteins is not yet understood. In vitro, cholesterol is known to affect micelle physical properties and has been found to interact directly with some membrane proteins. Previous studies of a G protein-coupled receptor (GPCR), human adenosine A2A receptor (hA2AR), purified using the detergent dodecylmaltoside (DDM) showed that the presence of a cholesterol analog was critical for ligand binding and the structural stability of the receptor. Here, we present biophysical characterization and ligand binding studies of the hA2AR purified using a short chain lipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC), which indicate that cholesterol is not needed to maintain the functionality and stability of the hA2AR in this lipid system. Differences in stability and ligand binding affinity are observed when the receptor is purified using these detergent and lipid systems, emphasizing the importance of the solubilization environment on receptor functionality. We will highlight the use of fluorescence polarization in characterizing this stability, for example, following protein storage or lipid addition.

Chemical semi-IPN hydrogels for the removal of adhesives from canvas paintings

Semi-interpenetrating (IPN) poly (2-hydroxy- ethyl methacrylate)/polyvinylpyrrolidone hydrogels were synthesized and used for the removal of adhesives from the back of canvas paintings. The high water retention capa- bility and the specific mechanical properties of these gels allow the safe cleaning of water-sensitive artifacts using water-based detergent systems. The cleaning action is limited to the contact area and layer-by-layer removal is achieved while avoiding water spreading and absorption within water-sensitive substrates, which could lead, for example, to paint detachment. The use of these chemical gels also avoids leaving residues over the treated surface because the gel network is formed by covalent bonds that provide high mechanical strength. In this contribution, the physicochemical characterization of semi-IPN chemical hydrogels is reported. The successful application of an o/w microemulsion confined in the hydrogel for the removal of adhesives from linen canvas is also illustrated.

Innovative Hydrogels Based on Semi-Interpenetrating p(HEMA)/PVP Networks for the Cleaning of Water-Sensitive Cultural Heritage Artifacts

Water-based detergent systems offer several advantages, over organic solvents, for the cleaning of cultural heritage artifacts in terms of selectivity and gentle removal of grime materials or aged varnish, which are known to alter the readability of the painting. Unfortunately, easel paintings present specific characteristics that make the usage of water-based systems invasive. The interaction of water with wood or canvas support favors mechanical stresses between the substrate and the paint layers leading to the detachment of the pictorial layer. In order to avoid painting loss and to ensure a fine control (layer by layer) of grime removal, water-based cleaning systems have been confined into innovative chemical hydrogels, specifically designed for cleaning water-sensitive cultural heritage artifacts. The synthesized hydrogels are based on semi-interpenetrating chemical poly(2-hydroxyethyl methacrylate)/poly(vinylpyrrolidone) networks with suitable hydrophilicity, water retention properties, and required mechanical strength to avoid residues after the cleaning treatment. Three different compositions were selected. Water retention and release properties have been studied by quantifying the amount of free and bound water (from differential scanning calorimetry); mesoporosity was obtained from scanning electron microscopy; microstructure from small angle X-ray scattering. To demonstrate both the efficiency and versatility of the selected hydrogels in confining and modulating the properties of cleaning systems, a representative case study is presented.

EPR Spectra of Spin-Labeled OmpA Reflect Both Protein Dynamics and Label Configuration in Membrane Mimetic Systems

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a powerful tool that can provide information on the structure and dynamics of proteins. It has been used successfully in both water soluble and membrane proteins. Because SDSL has no molecular weight limitation, it can be performed under physiological conditions and can give data on systems inaccessible to techniques such as solution NMR and X-ray crystallography. The EPR spectra of labeled sites in the outer membrane protein OmpA are highly dependent upon environment, and generally the spectra indicate that labels (R1 side chain) have less motional averaging in the presence of lipids than in detergent systems.

Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation

An understanding of the folding states of α-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in nuclear magnetic resonance spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation, we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.

Effects of glycerol and urea on micellization, membrane partitioning and solubilization by a non-ionic surfactant

We have studied the effect of two cosolvents, urea and glycerol, on the association and interactions of a surfactant, octaethyleneglycol dodecyl ether (C12EO8) and a phospholipid (POPC). We have measured the CMC, the partition coefficient, the effective mole fractions Xesat and Xesol at the onset and completion of the membrane-to-micelle transition (membrane solubilization), and the enthalpies of transfer of surfactant by ITC. Changes in membrane order and dynamics were characterized by time-resolved fluorescence anisotropy measurements of DPH, and micelle sizes and clouding by light scattering. The cosolvents have complex effects that are not governed by the well-known ‘salting in’ or ‘salting out’ effects on the solubility alone. Instead, urea and glycerol alter also the intrinsic curvature (‘effective molecular shape’) of the detergent and the order and packing of the membrane. These curvature effects have an unusual enthalpy/entropy balance and are not additive for lipid and detergent. The results shed light on the phenomena governing lipid–detergent systems in general and have a number of implications for the use of cosolvents in membrane protein studies.

Energetics of Glycophorin A Dimerization in Mammalian Plasma Membranes

Quantitative measurements of protein interaction strengths are crucial for describing signaling networks and predicting cellular responses to environmental stimuli. Of all interactions between biological macromolecules, interactions between membrane proteins are the least characterized, and their strength is often measured in model detergent and lipid systems that poorly mimic the complex biological membrane. Measurements in models systems, however, are not likely to yield accurate predictions, because interactions in the native cellular environment occur within the context of a crowded system.Here we explore the utility of plasma membrane-derived vesicles as a model crowded environment for quantitative characterization of membrane protein interactions in mammalian membranes. In particular, we study the dimerization energetics of Glycophorin A (GpA), the primary sialoglycoprotein of human erythrocyte membranes, using the “quantitative imaging Foster resonance energy transfer (QI-FRET)” method. We determine the FRET efficiency, and the donor and the acceptor concentrations in single plasma membrane-derived vesicles loaded with GpA. These measurements yield, for the first time, the free energy of GpA dimerization in mammalian membranes. Supported by NSF MCB-0718841.Li E, Placone J, Merzlyakov M, Hristova K (2008) Quantitative measurements of protein interactions in a crowded cellular environment. Anal Chem 80: 5976-5985

CD and EPR Structural Studies on the KCNE1 Protein in a Lipid Bilayer

KCNE1, also known as minK, is a membrane protein responsible for modulating the KCNQ1 voltage-gated potassium ion channel in the human heart. Previous in vivo electrophysiological studies have shown that KCNQ1 loses its functionality in the absence of KCNE1, showing that KCNE1 is an essential protein for proper heart function (Sanders et al., Biochemistry 2007 46:11459-11472). Though KCNE1 has been extensively studied in micelle detergent systems, little work has been done to study the protein in an actual lipid bilayer-membrane system. Our current research uses biophysical techniques such as circular dichrosim (CD) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy to characterize and compare KCNE1 proteins in various micelle and lipid bilayer environments using both qualitative and quantitative methods. Our CD spectroscopy experiments have shown that KCNE1 undergoes a change in secondary structure when removed from a micelle environment and placed in a lipid bilayer. We have used EPR spectroscopy to show that the dynamic properties of KCNE1 also change when taken out of micelles and inserted into lipid bilayers. Calculations have been done to quantify these differences in the structural and dynamic properties observed for KCNE1 in micelles and lipid bilayers.

Polar residues in transmembrane helices can decrease electrophoretic mobility in polyacrylamide gels without causing helix dimerization

There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as a membrane mimetic system. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents.