Unveiling the Hidden World of Membrane Proteins
On the cell surface, proteins play a crucial role in many bodily processes and are linked to various diseases. However, they are difficult to study due to their low numbers and water-repelling nature. Our team has created peptidiscs that mimic cell membranes, making it easier to study these proteins using advanced techniques. In a recent study, we used this method to identify hundreds of cell-surface proteins in different mouse organs and understand their abundance. This has improved our understanding of how these proteins work in specific organs. Our work has the potential to advance biomarker exploration and other biomedical research by shedding light on these mysterious proteins.

Membrane proteins play critical roles at the cell surface, and their misfunction is a hallmark of many human diseases. A precise evaluation of the plasma -membrane proteome is, therefore, essential for cell biology and for discovering novel biomarkers and therapeutic targets. However, the low abundance of this proteome relative to soluble proteins makes it difficult to characterize, even with the most advanced proteomics technologies. Here, we apply the peptidisc membrane mimetic to purify the cell membrane proteome. Using the HeLa cell line as a reference, the peptidisc captures over 500 integral membrane proteins, with half annotated to the plasma membrane. We extend the method to compare two pancreatic cell lines. Furthermore, since the method stabilizes membrane proteins in a water-soluble state, proteins in the library can be purified.

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.

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 β-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.

The 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 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.

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5′-triphosphate (ATP)–binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain.

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.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroidesreaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coliproteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy.  The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins.  Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a “one-size-fits-all” approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss.

We apply the peptidisc for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we find the peptidisc library very useful for identifying transient and novel protein interactions. Most of these interactions are largely undetected by standard detergent-based purification. The peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions. 

SecA–SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. A landmark previous study reported that ATP binding to SecA triggers a “power stroke,” resulting in forward movement of a polypeptide segment into the mouth of the SecYEG channel. Following ATP hydrolysis, when ADP is still bound to the enzyme, SecA adopts a conformation allowing the polypeptide to slide across the SecYEG channel until the next ATP- binding event. Instead, SecA rather operates as a “Brownian ratchet,” allowing substrates to passively diffuse within the SecY channel while the enzyme is in an ADP-state.

Obtaining membrane bound proteins in a stable non-aggregated state remains a problem in the scientific community. Previous methods of using detergent micelles (small amphipathic molecules) are quite often detrimental to protein structure and activity, in addition to interfering with downstream analytical methods. However, using multiple copies of an amphipathic bi-helical peptide (here after termed NSPr) wrapping around a target membrane protein allows for its solubilization, retention of 3D structure and protein activity. Therefore allowing membrane proteins to be studied in a solubilized state.

The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM…..

Nuclease colicins are antibacterial proteins produced by certain strains of E. coli to reduce competition from rival strains. How exactly colicins traverse the cell envelope is not understood, yet this knowledge is important for the design of new antibacterial therapies. In this report, we discover that the cytotoxic rRNAse domain of colicin E3 is sufficient to inhibit cell growth. There is high affinity interaction (Kd ~1-2μM) between the rRNAse domain and lipopolysaccharides (LPS) which is important to destabilize the secondary structure of the toxin, which is expectedly crucial for transport through the membrane. The effect of LPS on binding and unfolding of ColE3 may be indicative of a broader role of LPS for transport of colicins in general.

ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that chloride conductance occurring, to a degree that can be large enough to compromise cell viability. We conclude that ABC transporters must stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert transporter to channel.

ATP-binding cassette (ABC) transporters have evolved an ATP-dependent alternating-access mechanism to transport substrates across membranes. Despite important progress, especially in their structural analysis, it is still unknown how the substrate stimulates ATP hydrolysis, the hallmark of ABC transporters. In this study, we measure the ATP turnover cycle of MalFGK2 in steady and pre-steady state conditions. We show that (i) the basal ATPase activity of MalFGK2 is very low because the cleavage of ATP is rate-limiting, (ii) the binding of open-state MalE to the transporter induces ATP cleavage but leaves release of Pi limiting, and (iii) the additional presence of maltose stimulates release of Pi, and therefore increases the overall ATP turnover cycle. We conclude that open-state MalE stabilizes MalFGK2 in the outward-facing conformation until maltose triggers return to the inward-facing state for substrate and Pi release. This concerted action explains why ATPase activity of MalFGK2 depends on maltose, and why MalE is essential for transport.

Some metals and vitamins are critical for bacterial cell growth but their availability in the environment is limited. Gram-negative bacteria have evolved a set of outer membrane receptors to efficiently captureand transport these nutrients. Ferricrocin binds to FhuA to trigger the exposure of the TonB box to the periplasm. After recognition by TonB, the plug domain is pulled out of the β-barrel to allow the release of ferricrocin. Reconstitution of FhuA into nanodiscs binds TonB with a much lower affinity (~200 nM) than previously reported in detergent (~20 nM). Significantly, the binding critically depends on ferricrocin. FhuA in nanodiscs also forms a high-affinity binding site for colicin M.

The complex MalFGK2 hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK2 and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state.

A conserved protein-conducting channel, SecYEG in bacteria or Sec61p in eukaryotes, is the point of convergence of post- and co-translational protein targeting pathways and mediates the translocation or integration of newly synthesized proteins. Extensive work on the Sec pathway showed that SecYEG and pre-proteins stimulate SecA’s ATPase activity and activate it to drive the translocation of pre-proteins into the periplasm. During targeting, SRP and FtsY form a heterodimer in which both proteins undergo a series of conformational changes, including the early, closed, and activated states, that culminate in reciprocal GTPase activation.

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane.

Like the conductor of an orchestra, the Sec protein translocation channel is the platform needed to bring together the many different players required for the constitutive and obligatory process of protein transport. This conserved membrane channel, termed SecY in bacteria and Sec61 in eukaryotes, creates a ubiquitous protein-conducting pathway by which thousands of newly synthesized polypeptides make their way through the lipid bilayer. The channel is not a simple passive pore, however; it displays remarkable complexity by interacting with numerous soluble partners, including SecA, Syd, FtsY and the ribosome in bacteria.

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring-structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion-conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring-structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.