Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.

ß-barrel membrane proteins play a crucial role in bacterial pathogenesis and antibiotic resistance, making them a prime focus for the development of new antibiotics and therapeutics. However, their inherent hydrophobic nature and limited presence pose challenges for their high-throughput characterization using conventional methods. In this context, we present a simple but efficacious approach using peptidisc, a membrane mimetic, to overcome the low abundance and hydrophobicity of these proteins. Our methodology, illustrated here using Escherichia coli (E. coli) as a model organism, covers the entire process from outer membrane fraction preparation to data analysis. This detailed protocol outlines the purification of a diverse collection of ß-barrel membrane proteins, rendering them water-soluble and readily amenable to mass spectrometry and downstream drug screening strategies.

Characterization of membrane protein interactions by peptidisc-mediated mass photometry.

Membrane proteins perform numerous critical functions in the cell, making many of them primary drug targets. However, their preference for a lipid environment makes them challenging to study using established solution-based methods. Here, we show that peptidiscs, a recently developed membrane mimetic, provide an ideal platform to study membrane proteins and their interactions with mass photometry (MP) in detergent-free conditions. The mass resolution for membrane protein complexes is similar to that achievable with soluble proteins owing to the low carrier heterogeneity. Using the ABC transporter BtuCD, we show that MP can quantify interactions between peptidisc-reconstituted membrane protein receptors and their soluble protein binding partners. Using the BAM complex, we further show that MP reveals interactions between a membrane protein receptor and a bactericidal antibody. Our results highlight the utility of peptidiscs for membrane protein characterization in detergent-free solution and provide a rapid and powerful platform for quantifying membrane protein interactions.

Recent advances in membrane mimetics for membrane protein research.

Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and biophysical methods, membrane proteins must first be extracted from cellular membranes - often using detergents. Detergent-extracted membrane proteins are amenable to analysis by structural, biochemical, and biophysical techniques. In certain cases, however, detergents can disturb native protein conformations and/or biological activity. This has led to the development of membrane mimetics, which stabilize membrane proteins in a native membrane-like environment that is water-soluble and detergent-free. This review provides an overview of recent developments in the membrane mimetic field, with a focus on nanodiscs, Saposin lipid nanoparticles (SapNPs), peptidiscs, and SMA lipid particles (SMALPs) - and highlights their utility for supporting biophysical, biochemical, and structural characterization of membrane proteins and complexes.

A Dual Detergent Strategy to Capture a Bacterial Outer Membrane Proteome in Peptidiscs for Characterization by Mass Spectrometry and Binding Assays.

The outer membrane of Gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics, and therefore is a prime target for antimicrobial discovery. To facilitate the discovery efforts, a precise knowledge of the outer membrane proteome, and possible variations during pathogenesis, is important. Characterization of the bacterial outer membrane remain challenging, however, and low throughput, due to the high hydrophobicity and relatively low abundance of this cell compartment. Here we adapt our peptidisc-based method to selectively isolate the outer membrane proteome before analysis by mass spectrometry. Using a dual detergent membrane solubilization approach, followed by protein purification in peptidiscs, we capture over 70 outer membrane proteins, including 26 integral ß-barrels and 26 lipoproteins. Many of these proteins are present at high peptide intensities, indicative of a high abundance in the library sample. We further show that the isolated outer membrane proteome can be employed in downstream ligand-binding assays. This peptidisc library made of outer membrane proteins may therefore be useful to systematically survey other bacterial outer membrane proteomes, but also as a nanoparticle format able to support the discovery of next-generation antimicrobials. Data are available via ProteomeXchange identifier PXD036749.

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize, and Purify a Detergent-Sensitive Membrane Protein Assembly.

The peptidisc membrane mimetic enables global reconstitution of the bacterial membrane proteome into water-soluble detergent-free particles, termed peptidisc libraries. We present here a method that combines peptidisc libraries and chromosomal-level gene tagging technology with affinity purification and mass spectrometry (AP/MS) to stabilize and identify fragile membrane protein complexes that exist at native expression levels. This method circumvents common artifacts caused by bait protein overproduction and protein complex dissociation due to lengthy exposure to detergents during protein isolation. Using the Escherichia coli Sec system as a case study, we identify an expanded version of the translocon, termed the HMD complex, consisting of nine different integral membrane subunits. This complex is stable in peptidiscs but dissociates in detergents. Guided by this native-level proteomic information, we design and validate a procedure that enables purification of the HMD complex with minimal protein dissociation. These results highlight the utility of peptidiscs and AP/MS to discover and stabilize fragile membrane protein assemblies. Data are available via ProteomeXchange with identifier PXD032315.

Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.

His-Tagged Peptidiscs Enable Affinity Purification of the Membrane Proteome for Downstream Mass Spectrometry Analysis.

Characterization of the integral membrane proteome by mass spectrometry (MS) remains challenging due its high complexity and inherent insolubility. In a typical experiment, the cellular membranes are isolated, the proteins are solubilized and fractionated, and the detergent micelles are removed before MS analysis. Detergents are not compatible with mass spectrometry, however, and their removal from biological samples often results in reduced protein identification. As an alternative to detergents, we recently developed the peptidisc membrane mimetic, which allows entrapment of the cell envelope proteome into water-soluble particles, termed a "peptidisc library". Here, we employ a His-tagged version of the peptidisc peptide scaffold to enrich the reconstituted membrane proteome by affinity chromatography. This purification step reduces the sample complexity by depleting ribosomal and soluble proteins that often cosediment with cellular membranes. As a result, the peptidisc library is enriched in low-abundance membrane proteins. We apply this method to survey changes in the membrane proteome upon depletion of the SecDFyajC complex, the ancillary subunit of the Sec translocon. In the depleted strain, we detect increased membrane localization of the motor ATPase SecA, along with increased levels of an unannotated inner membrane protein, YibN. Together, these results demonstrate the utility of the peptidisc for global purification of membrane proteins and for monitoring change in the membrane proteome.

Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries.

Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a 'one-size fits all' membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution.

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.