Membrane Lipid-protein Interactions Research Articles

Native mass spectrometry of membrane protein-lipid interactions in different detergent environments.

Native mass spectrometry (MS) is revealing the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer; however, commonly used detergents for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility for characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable characterization of lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find protein-lipid interactions are not only protein-dependent but can also be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery, as well as biochemical and structural investigations.

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry.

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and MS. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the Escherichia coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extracts, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extracts, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid-binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

Interplay between membrane proteins and membrane protein-lipid pertaining to plant salinity stress.

High salinity in agricultural lands is one of the predominant issues limiting agricultural yields. Plants have developed several mechanisms to withstand salinity stress, but the mechanisms are not effective enough for most crops to prevent and persist the salinity stress. Plant salt tolerance pathways involve membrane proteins that have a crucial role in sensing and mitigating salinity stress. Due to a strategic location interfacing two distinct cellular environments, membrane proteins can be considered checkpoints to the salt tolerance pathways in plants. Related membrane proteins functions include ion homeostasis, osmosensing or ion sensing, signal transduction, redox homeostasis, and small molecule transport. Therefore, modulating plant membrane proteins' function, expression, and distribution can improve plant salt tolerance. This review discusses the membrane protein-protein and protein-lipid interactions related to plant salinity stress. It will also highlight the finding of membrane protein-lipid interactions from the context of recent structural evidence. Finally, the importance of membrane protein-protein and protein-lipid interaction is discussed, and a future perspective on studying the membrane protein-protein and protein-lipid interactions to develop strategies for improving salinity tolerance is proposed.

Phospholipid Preparations to Characterize Protein-Lipid Interactions In Vitro.

The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein-lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs.

The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter trimer (AmtB) and aquaporin Z (AqpZ) selectively remodel their local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate the membrane protein with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent extraction as an orthogonal approach to study membrane protein remodeling of lipids in the nanodisc with native MS. Our results showed that Triton X-100 and lauryldimethylamine oxide retain lipid selectivity that agrees with gas-phase ejection, but C8E4 distorts some preferential lipid interactions. Both approaches reveal that AmtB has a few selective binding sites for phosphatidylcholine (PC) lipids, is selective for binding phosphatidylglycerols (PG) overall, and is nonselective for phosphatidylethanolamines (PE). In contrast, AqpZ prefers either PC or PG over PE and prefers PC over PG. Overall, these experiments provide a picture of how membrane proteins bind different lipid head groups in the context of mixed lipid bilayers.

Entropy in the Molecular Recognition of Membrane Protein-Lipid Interactions.

Understanding the molecular driving forces that underlie membrane protein-lipid interactions requires the characterization of their binding thermodynamics. Here, we employ variable-temperature native mass spectrometry to determine the thermodynamics of lipid binding events to the human G-protein-gated inward rectifier potassium channel, Kir3.2. The channel displays distinct thermodynamic strategies to engage phosphatidylinositol (PI) and phosphorylated forms thereof. The addition of a 4'-phosphate to PI results in an increase in favorable entropy. PI with two or more phosphates exhibits more complex binding, where lipids appear to bind two nonidentical sites on Kir3.2. Remarkably, the interaction of 4,5-bisphosphate PI with Kir3.2 is solely driven by a large, favorable change in entropy. Installment of a 3'-phosphate to PI(4,5)P2 results in an altered thermodynamic strategy. The acyl chain of the lipid has a marked impact on binding thermodynamics and, in some cases, enthalpy becomes favorable.

Lipid-protein interactions in virus assembly and budding from the host cell plasma membrane.

Lipid enveloped viruses contain a lipid bilayer coat that protects their genome to help facilitate entry into the new host cell. This lipid bilayer comes from the host cell which they infect. After viral replication, the mature virion hijacks the host cell plasma membrane where it is then released to infect new cells. This process is facilitated by the interaction between phospholipids that make up the plasma membrane and specialized viral matrix proteins. This step in the viral lifecycle may represent a viable therapeutic strategy for small molecules that aim to block enveloped virus spread. In this review, we summarize the current knowledge on the role of plasma membrane lipid–protein interactions on viral assembly and budding.

Lipid head group adduction to soluble proteins follows gas-phase basicity predictions: Dissociation barriers and charge abstraction

Native mass spectrometry analysis of membrane proteins has yielded many useful insights in recent years with respect to membrane protein-lipid interactions, including identifying specific interactions and even measuring binding affinities based on observed abundances of lipid-bound ions after collision-induced dissociation (CID). However, the behavior of non-covalent complexes subjected to extensive CID can in principle be affected by numerous factors related to gas-phase chemistry, including gas-phase basicity (GB) and acidity, shared-proton bonds, and other factors. A recent report from our group showed that common lipids span a wide range of GB values. Notably, phosphatidylcholine (PC) and sphingomyelin lipids are more basic than arginine, suggesting they may strip charge upon dissociation in positive ion mode, while phosphoserine lipids are slightly less basic than arginine and may form especially strong shared-proton bonds. Here, we use CID to probe the strength of non-specific gas-phase interactions between lipid head groups and several soluble proteins, used to deliberately avoid possible physiological protein-lipid interactions. The strengths of the protein-head group interactions follow the trend predicted based solely on lipid and amino acid GBs: phosphoserine (PS) head groups form the strongest bonds with these proteins and out-compete the other head groups studied, while glycerophosphocholine (GPC) head groups form the weakest interactions and dissociate carrying away a positive charge. These results indicate that gas-phase thermochemistry can play an important role in determining which head groups remain bound to protein ions with native-like structures and charge states in positive ion mode upon extensive collisional activation.

Developing Nanodisc-ID for label-free characterizations of membrane proteins

Membrane proteins (MPs) influence all aspects of life, such as tumorigenesis, immune response, and neural transmission. However, characterization of MPs is challenging, as it often needs highly specialized techniques inaccessible to many labs. We herein introduce nanodisc-ID that enables quantitative analysis of membrane proteins using a gel electrophoresis readout. By leveraging the power of nanodiscs and proximity labeling, nanodisc-ID serves both as scaffolds for encasing biochemical reactions and as sensitive reagents for detecting membrane protein-lipid and protein-protein interactions. We demonstrate this label-free and low-cost tool by characterizing a wide range of integral and peripheral membrane proteins from prokaryotes and eukaryotes.

Impact of membrane protein-lipid interactions on formation of bilayer lipid membranes on SAM-modified gold electrode

In protein film electrochemistry for redox-active membrane proteins, the reconstitution of protein-tethered bilayer lipid membranes (ptBLMs) can maximize their functionality and stability at electrolyte/electrode interfaces. To understand the impact of protein–lipid interactions on the ptBLM formation, we tracked the reconstitution process of a lipid bilayer in the presence or the absence of a transmembrane enzyme of cytochrome c-dependent nitric oxide reductase (cNOR) on a mixed self-assembled monolayer (SAM)/Au electrode. Time-resolved surface-enhanced infrared absorption (SEIRA) spectroscopy and electrochemical measurements revealed that the protein–lipid interaction affected the formation kinetics for the phospholipid bilayer and the electrocatalytic stability of the cNOR-modified electrode but not the fluidity of the phospholipid bilayer.

Lipid-Dependent Ensemble of Conformational Substates in Rhodopsin Activation Studied by FTIR and UV-Visible Spectroscopy

G protein-coupled receptors (GPCRs) transmit signals across lipid membranes where rhodopsin is an important prototype. Here we address the role of soft matter in GPCR function for this class of integral membrane proteins [1,2]. Our hypothesis is the process involves an ensemble of activated states affected by membrane protein-lipid bilayer interactions [1]. Rhodopsin was investigated in lipid membranes (native disk membranes, POPC, DOPC) by Fourier transform infrared (FTIR) and UV-visible spectroscopy. Application of different spectroscopic methods together with pH titration at various temperatures allowed us to identify distributions of states in the MI-to-MII activating equilibrium and determine equilibrium constants and thermodynamic parameters of the transitions. Analysis of pH titration curves indicated that more than one activated state exists [3] supporting the concept of rhodopsin as a dynamically activated receptor. Furthermore, temperature changes had a crucial effect on rhodopsin activation enabling coexistence and simultaneous observation of multiple inactive and active states. Variation of the lipid composition indicated the POPC bilayer noticeably shifted the equilibrium to the inactive MI state [1] while the DOPC behavior was similar to native membranes. To improve data reduction, we analyzed the MI-to-MII equilibrium for the whole range of FTIR spectra where changes were observed between the MI and MII states. The results show the distribution of pKa and alkaline end-point values for all temperatures and different lipid compositions, indicating that activation of rhodopsin yields a conformational ensemble (collectively designated as MII state). We hypothesize that collective large-scale helical fluctuations in the metarhodopsin equilibrium are coupled to activation of transducin in visual signaling. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] U. Chawla et al. (2020) Angew. Chem. Int. doi.org/10.1002/anie.202003342. [3] M. Mahalingam et al. (2008) PNAS 105, 17795-17800.

Proteoliposomes for Studying Lipid-protein Interactions in Membranes in vitro.

Lipids in biomembranes can control the structure and, therefore, the functionality of membrane-embedded protein complexes. Unraveling how the lipid composition determines the mode of operation of membrane proteins provides mechanistic insights into their functionality. We applied a proteoliposome technique for studying how proteins function in biomembranes. The incorporation of isolated membrane proteins in preformed liposomes made from a well-defined lipid composition (proteoliposomes) is a powerful tool for studying lipid-protein interactions. Over several decades, the proteoliposome technique was employed for many different membrane proteins. Recently, it was recognized that different lipid compositions control the light-harvesting functionality of the major photosynthetic light-harvesting complex II (LHCII) isolated from plant thylakoid membranes in vitro. This technique allows systematic examination of the role of so-called non-bilayer lipids on light-harvesting characteristics of LHCII. This protocol describes the isolation of LHCII from leaves and details a four-step procedure to incorporate the detergent-solubilized membrane protein in large unilamellar vesicles (LUV). The protocol was optimized to ensure a very high lipid/protein ratio, designed to specifically examine lipid-protein interactions by minimizing LHCII aggregation. The procedure provides structurally and functionally highly intact LHCII in a detergent-free lipid bilayer with a defined composition.

Nanodiscs and mass spectrometry: Making membranes fly

Cells are surrounded by a protective lipid bilayer membrane, and membrane proteins in the bilayer control the flow of chemicals, information, and energy across this barrier. Many therapeutics target membrane proteins, and some directly target the lipid membrane itself. However, interactions within biological membranes are challenging to study due to their heterogeneity and insolubility. Mass spectrometry (MS) has become a powerful technique for studying membrane proteins, especially how membrane proteins interact with their surrounding lipid environment. Although detergent micelles are the most common membrane mimetic, nanodiscs are emerging as a promising platform for MS. Nanodiscs, nanoscale lipid bilayers encircled by two scaffold proteins, provide a controllable lipid bilayer for solubilizing membrane proteins. This Young Scientist Perspective focuses on native MS of intact nanodiscs and highlights the unique experiments enabled by making membranes fly, including studying membrane protein-lipid interactions and exploring the specificity of fragile transmembrane peptide complexes. It will also explore current challenges and future perspectives for interfacing nanodiscs with MS.

Gas-Phase Protonation Thermodynamics of Biological Lipids: Experiment, Theory, and Implications.

Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein-lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein-lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the "Extended" Kinetic Method and ab initio computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, ΔG for deprotonation of the protonated base) and proton affinities (PA, ΔH for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (ΔG and ΔH for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein-lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein-lipid complexes.

Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR.

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σN,tail =-1.8±0.1 s-1 M-1 and σN,head =-0.5±0.3 s-1 M-1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.

Characterizing the Locus of a Peripheral Membrane Protein-Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli.

Quantitative characterization of the strength of peripheral membrane protein-lipid bilayer interactions is fundamental in the understanding of many protein targeting pathways. SecA is a peripheral membrane protein that plays a central role in translocating precursor proteins across the inner membrane of E. coli. The membrane binding activity of the extreme N-terminus of SecA is critical for translocase function. Yet, the mechanical strength of the interaction and the kinetic pathways that this segment of SecA experiences when in proximity of an E. coli polar lipid bilayer has not been characterized. We directly measured the N-terminal SecA-lipid bilayer interaction using precision single molecule atomic force microscope (AFM)-based dynamic force spectroscopy. To provide conformational data inaccessible to AFM, we also performed all-atom molecular dynamics simulations and circular dichroism measurements. The N-terminal 10 amino acids of SecA have little secondary structure when bound to zwitterionic lipid head groups, but secondary structure, which rigidifies the lipid-bound protein segment, emerges when negatively charged lipids are present. Analysis of the single molecule protein-lipid dissociation data converged to a well-defined lipid-bound-state lifetime in the absence of force, τ0lipid = 0.9 s, which is well separated from and longer than the fundamental time scale of the secretion process, defined as the time required to translocate a single amino acid residue (∼50 ms). This value of τ0lipid is likely to represent a lower limit of the in vivo membrane-bound lifetime due to factors including the minimal system employed here.

Measuring Membrane Protein-Lipid Interactions in Nanodiscs with Native Mass Spectrometry

Native mass spectrometry (MS), which uses nondenaturing ionization to preserve noncovalent complexes for mass analysis, has proven to be powerful for characterizing membrane protein complex stoichiometry and interactions. Membrane proteins are traditionally solubilized using detergent micelles prior to native MS. Collisional activation is employed in the mass spectrometer to remove bound detergent and yield the membrane protein, potentially in complex with bound lipids. However, this method of solubilization can be problematic as detergents may distort the native-like structure and function of membrane proteins. Furthermore, the collisional activation required for detergent removal can lead to the loss of noncovalent interactions within fragile complexes. Lipoprotein nanodiscs provide a more natural lipid bilayer environment for delivering membrane proteins for native MS, but previous measurements have been limited by the moderate stability of nanodiscs in the gas phase. It is difficult to preserve the intact membrane protein-nanodisc complex for native MS but also difficult to efficiently eject membrane proteins from nanodiscs. To tune the stability of nanodiscs, we used chemical additives known to modulate the charge acquired during electrospray ionization (ESI). Two membrane proteins, the trimeric ammonium transporter AmtB and tetrameric aquaporin ApqZ, were reconstituted into nanodiscs for native MS. Employing charge manipulation reagents and different ionization polarities allowed us to either capture the intact membrane protein-nanodisc complex or eject the membrane protein with few bound lipids. We are currently investigating the selectivity of lipid binding to membrane proteins ejected from nanodiscs of mixed lipid compositions. Preliminary results show that distinct lipid shells corresponding to different types of protein-lipid interactions are preferentially enriched in either zwitterionic or anionic lipids. Ultimately, we expect our novel methods combining native MS and nanodiscs will provide unique insights into membrane protein-lipid interactions in model lipid bilayers.

Insights Into Membrane Protein-Lipid Interactions from Free Energy Calculations

Insights Into Membrane Protein-Lipid Interactions from Free Energy Calculations

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.