Methods Mol. Biol. [JOURNAL]
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200 papers
Solution NMR Spectroscopy for the Determination of Structures of Membrane Proteins in a Lipid Environment.
Pandey R, Arora A
Methods Mol Biol · 2026 · PMID 42156694 · Publisher ↗
NMR spectroscopy has substantially benefited from the recent technical advances to maintain its position as an eminent technique for the characterization of the solution structures and dynamics of membrane proteins at th...
NMR spectroscopy has substantially benefited from the recent technical advances to maintain its position as an eminent technique for the characterization of the solution structures and dynamics of membrane proteins at the level of atomic resolution. NMR offers a combination of several versatile strategies, for example, choice of molecular assemblies, like micelles, bicelles, and nanodiscs, with appropriate deuterated or non-deuterated detergents and phospholipids; temperature, and ionic strength; isotope labeling with H, C, N, with or without protonation of Ala, Met, Ile (δ1), Leu, and Val methyl protons; combinatorial labeling or unlabeling of specific amino acids; TROSY based-, non-uniform sampling (NUS) based-, BEST, and other NMR experiments; measurement of residual dipolar couplings using stretched polyacrylamide gels or DNA nanotubes; and spin-labeling and paramagnetic relaxation enhancements (PRE). The right combinations of these strategies together with availability of high-field NMR spectrometers (upto 1.2 GHz H frequency) equipped with highly-sensitive cryogenically cooled-probes have allowed the perseverant investigator to successfully overcome several of the conventional pitfalls associated with the NMR technique and membrane proteins viz. low sensitivity, poor sample stability, spectral crowding, and a limited number of NOEs and other constraints for structure calculations. This has resulted in a steady growth in the number of successfully determined NMR structures of large and complex membrane proteins over the last two decades, and NMR spectroscopy in solution state is well-entrenched to hold its place amongst the various techniques used for the structure determination of an ever-larger number of membrane proteins.
Solid-State NMR Approaches to Study Protein Structure and Protein-Lipid Interactions.
Aisenbrey C, Salnikov ES, Raya J … +2 more , Michalek M, Bechinger B
Methods Mol Biol · 2026 · PMID 42156693 · Publisher ↗
Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phosph...
Solid-state NMR spectroscopy has been developed for the investigation of membrane-associated polypeptides and remains one of the few techniques to reveal high-resolution structural information in liquid-disordered phospholipid bilayers. While magic angle spinning solid-state NMR remains a popular approach yielding spectra that resemble those in solution, the investigation of static, uniaxially oriented samples provides an alternative concept that has been used to investigate the structure, dynamics, and topology of membrane polypeptides. Much of the previous solid-state NMR work has been developed and performed on peptides, but the technique is constantly expanding toward larger membrane proteins. Here, a number of protocols are presented describing, among others, the reconstitution of membrane proteins into oriented membranes, monitoring membrane alignment by P solid-state NMR spectroscopy, investigations of the protein by one- and two-dimensional N solid-state NMR and measurements of the lipid order parameters using H solid-state NMR spectroscopy. Using such methods, solid-state NMR spectroscopy has revealed a detailed picture of the ensemble of both lipids and proteins and their mutual interdependence in the bilayer environment.
Non-Linear Continuous Wave Electron Paramagnetic Resonance Spectroscopy for Investigation of Lipid-Protein Interactions in Membranes.
Páli T
Methods Mol Biol · 2026 · PMID 42156692 · Publisher ↗
This chapter describes how to utilize spin-spin interactions measured by electron paramagnetic resonance (EPR) to gain structural details of lipid-protein interactions in samples containing more than one type of paramagn...
This chapter describes how to utilize spin-spin interactions measured by electron paramagnetic resonance (EPR) to gain structural details of lipid-protein interactions in samples containing more than one type of paramagnetic molecules. Common in these methods is that certain types of continuous wave (CW) EPR signals (also called spectral displays) are recorded under partial microwave saturation, extending into the region of a non-linear dependence of some spectral features on the microwave power (hence, the name non-linear CW EPR), and spectral parameters are derived that are proportional to the longitudinal (T) relaxation time, which is in turn highly sensitive to spin-spin interaction between the paramagnetic molecules. Although powerful pulsed EPR techniques exist to measure T (and also for the transversal relaxation time, T) directly, the advantage of the methods described here is that they can be performed on the much cheaper and more widespread conventional CW EPR instruments. During the 1990s, we developed and refined T-sensitive nonlinear CW EPR techniques, which were then combined with paramagnetic quenching agents and doubly spin-labeled samples for distance measurements. In the following sections, first the concept of non-linear CW EPR will be introduced, then the common materials and methods will be described, followed by four example protocols and data analysis, and the chapter is concluded with useful notes. This chapter is incremental to the previous one in the sense that several experimental details are the same, such as the sources and types of spin-labeled lipid analogues, preparation of lipid-labeled bio- and model membranes, assaying lipid and protein content, sample geometry, basic instrument settings and processing of the EPR spectra (e.g., removal of the background and disturbing spectral components, normalization). Here, the focus is on the extra experimental and theoretical procedures required for measuring various types of spin-spin interactions and utilizing them for proximity relations in membranes. The example procedures and figures are illustrations, rather than exact reproductions of data from our previous works.
Electron Paramagnetic Resonance Spectroscopy of Spin-Labeled Lipids for Studying Lipid-Protein Interaction in Model and Biomembranes.
Páli T, Kóta Z
Methods Mol Biol · 2026 · PMID 42156691 · Publisher ↗
Spin label electron paramagnetic resonance (EPR) of lipid-protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin label EPR spectroscopy is the technique of choice...
Spin label electron paramagnetic resonance (EPR) of lipid-protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin label EPR spectroscopy is the technique of choice to characterize the protein-solvating lipid shell in its highly dynamic nature, because the EPR spectra of lipids that are spin-labeled close to the terminal methyl end of their acyl chains display two spectral components: those corresponding to lipids directly contacting the protein and those corresponding to lipids in the bulk fluid bilayer regions of the membrane. In this chapter, typical spin label EPR procedures are presented that allow the determination of the stoichiometry of interaction of spin-labeled lipids with the intramembranous region of membrane proteins or polypeptides, as well as the association constant of the spin-labeled lipid with respect to the host lipid. The experiments can be made on the most common, standard EPR spectrometers. The lipids giving rise to a so-called immobile spectral component in the EPR spectrum of such samples are identified as the motionally restricted first-shell lipids solvating membrane proteins in biomembranes. Stoichiometry and selectivity are directly related to the structure of the intramembranous sections of membrane-associated proteins or polypeptides [1] and can be used to study the state of assembly of such proteins in the membrane. Since these characteristics of lipid-protein interactions are discussed in detail in the literature (see [2, 3] for reviews), here we focus more on how to spin label model and biomembranes and how to measure and analyze the two-component EPR spectra of spin-labeled lipids in phospholipid bilayers that contain proteins or polypeptides. After a description of how to prepare spin-labeled model and native biological membranes, we present the reader with computational procedures for determining the molar fraction of motionally restricted lipids when both, one or none of the pure isolated-mobile or immobile-spectral components are available. With these topics, this chapter complements a previous methodological paper [4]. The interpretation of the data is discussed briefly, as well as other relevant and recent spin label EPR techniques for studying lipid-protein interactions, not only from the point of view of lipid chain dynamics.
EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers.
Bordignon E, Kucher S, Polyhach Y
Methods Mol Biol · 2026 · PMID 42156690 · Publisher ↗
Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological...
Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, e.g., in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or a few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity, and data interpretation; thus, it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book, and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques, providing information on side chain dynamics and water accessibility using nitroxide probes. An optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.
Sited-Directed Fluorescence Spectroscopy for Studies on the Folding and Insertion of Proteins in Lipid Bilayers and Model Membranes.
Nguyen L, Herwig S, Gerlach L … +3 more , Gholami S, Schürmann N, Kleinschmidt JH
Methods Mol Biol · 2026 · PMID 42156689 · Publisher ↗
Protein-lipid interactions are important for folding and membrane insertion of integral membrane proteins that are composed either of α-helical or of β-barrel structure in their transmembrane domains. While α-helical tra...
Protein-lipid interactions are important for folding and membrane insertion of integral membrane proteins that are composed either of α-helical or of β-barrel structure in their transmembrane domains. While α-helical transmembrane proteins fold cotranslationally that they are synthesized by a ribosome, β-barrel transmembrane proteins (β-TMPs) fold and insert post-translationally; in bacteria after translocation across the cytoplasmic membrane, in cell organelles of eukaryotes after import across the outer membrane of the organelle. β-TMPs unfold in aqueous solutions of high concentrations of chaotropic denaturants like urea and spontaneously refold upon denaturant dilution in the presence of preformed lipid bilayers. This facilitates studies on lipid interactions during folding into lipid bilayers. For several β-TMPs, the kinetics of folding has been reported as strongly dependent on protein-lipid interactions. The kinetics of adsorption/insertion and folding of β-TMPs can be monitored by fluorescence spectroscopy. These fluorescence methods are even more powerful when combined with site-directed mutagenesis for the preparation of mutants of a β-TMP that are site-specifically labeled with a fluorophore or a fluorophore and either a fluorescence quencher or fluorescence resonance energy acceptor. Single tryptophan or single cysteine mutants of the β-TMP allow the investigation of local protein-lipid interactions, at specific regions within the protein. To examine the structure formation of β-TMPs in a lipid environment, fluorescence spectroscopy has been used for double mutants of β-TMPs that contain a fluorescent tryptophan and a spin-label, covalently attached to a cysteine as a fluorescence quencher. The sites of mutation are selected in close proximity to another in the folded state of the β-TMP. In a folding experiment, the evolution of fluorescence or fluorescence quenching as a function of time at specific sites within the protein can provide important information on the folding mechanism of the β-TMP. Here, we report protocols to examine membrane protein folding for two β-TMPs in a lipid environment, the outer membrane protein A from Escherichia coli (OmpA) and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria.
Membrane Pore Formation by Peptides Studied by Fluorescence Techniques.
Tatulian SA, Kandel N
Methods Mol Biol · 2026 · PMID 42156688 · Publisher ↗
Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to prod...
Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to produce antimicrobial peptides to kill the pathogens by a similar membrane perforation mechanism. Furthermore, certain endogenous proteins or peptides kill the parent cells through membrane permeabilization. Analysis of the molecular details of membrane pore formation is often conducted using artificial systems, such as bilayer lipid membranes and synthetic peptides. This chapter describes two fluorescence-based methods to study peptide-induced membrane leakage. One method involves preparation of lipid vesicles loaded with a fluorophore (e.g., calcein or carboxyfluorescein) at a self-quenching concentration. If the externally added peptide forms relatively large pores (≥1 nm in diameter), the fluorophore leaks out and undergoes dequenching, resulting in a time-dependent increase in fluorescence. The other method is designed to monitor smaller pores (<1 nm in diameter). It involves the preparation of vesicles in a Ca-less buffer, containing a Ca-sensitive fluorophore, such as Quin-2. Removal of external Quin-2 by a desalting column and addition of an appropriate concentration of CaCl externally sequesters Quin-2 and Ca ions by the vesicle membrane. Addition of the pore-forming peptide to these vesicles results in membrane permeabilization, Ca influx and binding to Quin-2 monitored by an increase in Quin-2 fluorescence. In both cases, the kinetics of the increase of fluorescence and its equilibrium level allow quantitative analysis of the pore formation mechanism. Quantitative analysis of the membrane insertion depth of peptides using fluorescence quenching by brominated lipids is also presented in this chapter.
Fluorescence Correlation Spectroscopy to Examine Protein-Lipid Interactions in Membranes.
Betaneli V, Krohn JH, Mücksch J … +1 more , Schwille P
Methods Mol Biol · 2026 · PMID 42156687 · Publisher ↗
Fluorescence correlation spectroscopy (FCS) is a versatile technique to study membrane dynamics and protein-lipid interactions. It can provide information about diffusion coefficients, concentrations, and molecular inter...
Fluorescence correlation spectroscopy (FCS) is a versatile technique to study membrane dynamics and protein-lipid interactions. It can provide information about diffusion coefficients, concentrations, and molecular interactions of proteins and lipids in the membrane. These parameters allow the determination of protein partitioning into different lipid environments, the identification of lipid domains, and the detection of lipid-protein complexes on the membrane. During the last decades, FCS studies were successfully performed on model membrane systems as also on living cells, to characterize protein-lipid interactions. Recent developments of the method described here improved quantitative measurements on membranes and decreased the number of potential artifacts. The aim of this chapter is to provide the reader with the necessary information and some practical guidelines to perform FCS studies on artificial and cellular membranes.
A Guide to Tracking Single Membrane Proteins and Their Interactions in Supported Lipid Bilayers.
Taylor EL, Poudel KR, Brozik JA
Methods Mol Biol · 2026 · PMID 42156686 · Publisher ↗
The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single proteins in planar-supported biomimetic membranes. This chapter describes, in detail, the construction of a s...
The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single proteins in planar-supported biomimetic membranes. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) temperature control, (3) an alignment procedure, (4) a calibration procedure, and (5) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar-supported bilayers on hydrophilically treated borosilicate and quartz. These include (1) POPC bilayers, (2) POPC/PEG-PE cushioned bilayers, (3) POPC/PEG-PE cushioned bilayers on BSA passivated substrates, and (4) a cushioned biomimetic membrane of the endoplasmic reticulum (ER). A procedure for the detergent-mediated incorporation of the transmembrane protein 5HT (a serotonin receptor) is also described. It can be a starting point for other large, nonself-inserting transmembrane proteins. A procedure for the detergent-free incorporation of cytochrome P450 reductase (CPR) and cytochrome P450 enzymes (P450) into an ER biomimetic is also described. The final experimental section of this chapter details different procedures for data analysis, including (1) quantitative analysis of mean squared displacements from individually tracked proteins, (2) gamma distribution analysis of diffusion coefficients from a small ensemble of individually tracked proteins, (3) average mean squared displacement analysis, (4) Gaussian analysis of step-size distributions, (5) Arrhenius analysis of temperature-dependent data, (6) the determination of equilibrium constants from a step-size distribution, and (7) a perspective associated with the interpretation of single particle tracking data.
Förster Resonance Energy Transfer as a Tool for Quantification of Protein-Lipid Selectivity.
Loura LMS, Prieto M, Fernandes F
Methods Mol Biol · 2026 · PMID 42156685 · Publisher ↗
This chapter addresses the determination of protein-lipid selectivity, which is described as the preference of a protein for a specific type of lipid in its vicinity. Specifically, Förster resonance energy transfer (FRET...
This chapter addresses the determination of protein-lipid selectivity, which is described as the preference of a protein for a specific type of lipid in its vicinity. Specifically, Förster resonance energy transfer (FRET) methodologies are used for quantification of this effect. FRET enables the determination of biasing in the distribution of the lipid under study around the protein, as compared to its bulk membrane distribution, with advantages over alternative established approaches, such as electron spin resonance spectroscopy. The experiment can be carried out in either steady-state or time-resolved conditions. Formalisms valid for both situations are described in detail. They apply to both transmembrane-spanning and lipid/water interface-bound proteins of any size.
Analyzing Transmembrane Protein and Hydrophobic Helix Topography by Dual Fluorescence Quenching.
Caputo GA, London E
Methods Mol Biol · 2026 · PMID 42156684 · Publisher ↗
The location of fluorescent groups relative to the lipid bilayer can be evaluated using fluorescence quenchers embedded in the membrane and/or dissolved in aqueous solution. Quenching can be used to define the membrane t...
The location of fluorescent groups relative to the lipid bilayer can be evaluated using fluorescence quenchers embedded in the membrane and/or dissolved in aqueous solution. Quenching can be used to define the membrane topography of membrane proteins and individual membrane-embedded hydrophobic helices by combining it with the placement of fluorescent groups, including Trp, at defined sequence positions. This chapter briefly discusses various quenching methods for studies of membrane protein topography and provides detailed protocols for dual quencher analysis (DQA), a rapid, highly sensitive, and experimentally flexible approach in which the information gained from both a membrane-embedded and aqueous quencher is combined. The advantages of the DQA method include flexibility with regard to the bilayer compositions to which it can be applied, including membranes composed of lipids of varying headgroup and acyl chain compositions, as well as the ability to identify mixed populations of fluorophores residing at different depths within the bilayer.
Deep UV Resonance Raman Spectroscopy: Applications to Membrane Proteins and Peptides.
Tangtartharakul CB, Ruiz DG, Kim JE
Methods Mol Biol · 2026 · PMID 42156683 · Publisher ↗
UV resonance Raman (UVRR) spectroscopy is a vibrational technique that provides molecular insights with high selectivity. By tuning the Raman excitation wavelength across the deep UV region, from 207 to 228 nm, the Raman...
UV resonance Raman (UVRR) spectroscopy is a vibrational technique that provides molecular insights with high selectivity. By tuning the Raman excitation wavelength across the deep UV region, from 207 to 228 nm, the Raman signal of backbone or aromatic sidechains is selectively enhanced. This chapter describes the wavelength dependence of UVRR spectra and highlights the ability of 207 nm excitation to reveal the secondary structure of membrane-associated biomolecules.
Protein-Membrane Interactions Studied by Transmission and Total Internal Reflection FTIR Spectroscopy.
Tatulian SA
Methods Mol Biol · 2026 · PMID 42156682 · Publisher ↗
Fourier transform infrared (FTIR) spectroscopy has become one of the mainstream techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capab...
Fourier transform infrared (FTIR) spectroscopy has become one of the mainstream techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capability of providing the atomic-resolution structure of proteins or lipids, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. Sensitivity of vibrational frequencies to atomic masses has led to the development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, the structural features of membrane pore-forming proteins and peptides, and much more. The purpose of this chapter is to provide a practical guide for the analysis of protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, is presented.
Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Membrane Environment.
Matsuo K, Gekko K
Methods Mol Biol · 2026 · PMID 42156681 · Publisher ↗
Circular dichroism (CD) spectroscopy is a powerful tool for the secondary structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and...
Circular dichroism (CD) spectroscopy is a powerful tool for the secondary structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins under a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190 nm and producing information that cannot be obtained using conventional CD instruments. Combining SRCD data with bioinformatics, molecular dynamics simulations, and other polarization techniques, such as linear dichroism and fluorescence anisotropy, provides new insights into the conformational changes of proteins in a membrane environment.
Complex Structural Examination of Protein-Lipid Interactions with Neutron Scattering Techniques.
Mahmoudi N, Johnston H, Ayscough SE … +9 more , Hall S, Wacklin-Knecht H, Knowles TJ, Paracini N, Cárdenas M, Gröbner G, Lakey J, Heinrich F, Clifton LA
Methods Mol Biol · 2026 · PMID 42156680 · Publisher ↗
Neutron scattering has significant benefits for examining the structure of protein-lipid complexes. Cold neutrons are non-damaging and predominantly interact with the atomic nucleus, meaning that neutron beams can penetr...
Neutron scattering has significant benefits for examining the structure of protein-lipid complexes. Cold neutrons are non-damaging and predominantly interact with the atomic nucleus, meaning that neutron beams can penetrate deeply into samples, which allows for flexibility in the design of samples studied. Components within a complex can be individually resolved by leveraging the strong difference in neutron scattering length between protium ( , 99.984% natural abundance) and deuterium ( or D, 0.016%) namely through the mixing of HO and DO in the samples or by the deuterium labelling of the biomolecules. Thus, the relative distribution of components within a membrane can be elucidated. Using neutron scattering techniques lipid-protein complexes are most commonly studied using neutron reflectometry (NR) and small-angle neutron scattering (SANS). In this review, the methodologies to produce and examine a variety of model biological membrane systems using SANS and NR are detailed. These systems include supported lipid bilayers derived from vesicle dispersions or Langmuir-Blodgett deposition, tethered and floating bilayer systems, membrane protein-lipid complexes, and polymer wrapped lipid nanodiscs. The three key stages of any SANS/NR study on model membrane systems-sample preparation, data collection, and analysis-are described together with some background on the techniques themselves.
Protein Microarrays and Liposome: A Method for Studying Lipid-Protein Interactions.
Chen A, Herianto S, Chen CS … +1 more , Zhu H
Methods Mol Biol · 2026 · PMID 42156679 · Publisher ↗
Lipid-protein interactions provide valuable insights into the functions of both lipids and proteins. A novel and powerful technique for identifying these interactions involves the use of protein microarrays combined with...
Lipid-protein interactions provide valuable insights into the functions of both lipids and proteins. A novel and powerful technique for identifying these interactions involves the use of protein microarrays combined with liposomes. Liposomes are spherical vesicles encapsulated by phospholipid bilayers, into which specific lipids of interest can be incorporated. This allows liposomes to serve as effective tools for detecting lipid-protein interactions and enables the simultaneous analysis of thousands of proteins with a limited number of lipids in a single experiment. This chapter outlines the methodologies and protocols for employing protein microarray assays alongside liposome fabrication to investigate protein-lipid interactions. Additionally, recent research findings in this field are reviewed.
Probing the Annular Lipid Belt of Membrane Proteins Using Native Mass Spectrometry.
Kumar S, Laganowsky A
Methods Mol Biol · 2026 · PMID 42156678 · Publisher ↗
Membrane proteins are embedded within the biological membrane and encircled by an annular belt of lipids. Understanding the composition and dynamics of this annular belt is essential for advancing our knowledge of membra...
Membrane proteins are embedded within the biological membrane and encircled by an annular belt of lipids. Understanding the composition and dynamics of this annular belt is essential for advancing our knowledge of membrane proteins, which play critical roles in various physiological functions of living cells. Native mass spectrometry (MS) allows for the detection of intact membrane protein-lipid complexes in the gas phase. However, the conventional approach of solubilizing membrane proteins in detergents often interferes with the analysis of protein-lipid interactions due to the properties of the chosen detergent. In this study, we demonstrate how charge-reducing detergents and molecules, such as spermine, reduce the overall charge of membrane protein-lipid complexes, enabling the investigation of the annular lipid belt surrounding membrane proteins.
Imaging Lipid-Associated Macromolecular Structures by Optimized Negative-Staining Transmission Electron Microscopy.
Liu J, Zhang M, Ren G
Methods Mol Biol · 2026 · PMID 42156677 · Publisher ↗
Many macromolecules, including transmembrane proteins and apolipoproteins, interact with lipids and membranes and play crucial roles in diverse biological processes. Understanding macromolecule-lipid interactions at the...
Many macromolecules, including transmembrane proteins and apolipoproteins, interact with lipids and membranes and play crucial roles in diverse biological processes. Understanding macromolecule-lipid interactions at the structural level is essential for elucidating their functions and mechanisms. However, determining the structure of macromolecules, particularly proteins in their lipid-bound state, has traditionally been challenging for X-ray crystallography due to the conformational and compositional heterogeneity of macromolecule-lipid complexes. Transmission electron microscopy (TEM) offers a unique capability to directly visualizing individual macromolecular particles in the presence of lipid interactions. Among TEM techniques, negative staining (NS) is a fast and widely used approach for imaging macromolecules. However, conventional NS protocols often introduce artifacts in lipid-associated protein samples, such as rouleaux formation in lipoproteins, which can compromise structural interpretation. To address these limitations, Ren and colleagues developed an optimized negative staining (OpNS) protocol by refining earlier methods and validating the results against cryo-electron microscopy (cryo-EM) images of lipoproteins embedded in vitrified ice. This optimized protocol minimizes artifacts and produces "near-native" particle images with high quality. It enables more accurate structural analysis, particularly for three-dimensional (3D) reconstructions of single macromolecular particles without averaging, using individual-particle electron tomography (IPET). This improved protocol provides a robust, efficient, and reliable method for imaging macromolecules in their lipid-binding states, offering significant advances in understanding macromolecule-lipid interactions.
Analysis of the Kinetics of Folding and Insertion of β-Barrel Membrane Proteins by Gel-Electrophoresis.
Hoppe J, Marszal A, Herwig S … +2 more , Schüßler A, Kleinschmidt JH
Methods Mol Biol · 2026 · PMID 42156676 · Publisher ↗
Investigations on the mechanisms of folding and insertion of transmembrane proteins (TMPs) into lipid bilayers require an analysis of the time course of the structural changes of a TMP. Kinetic studies are essential for...
Investigations on the mechanisms of folding and insertion of transmembrane proteins (TMPs) into lipid bilayers require an analysis of the time course of the structural changes of a TMP. Kinetic studies are essential for the analysis of individual folding steps, for identification of folding intermediates and for obtaining activation energies and the rate-limiting steps of folding. For many β-barrel transmembrane proteins (β-TMPs), it has been shown that the folded, functional form can be separated from the unfolded, nonfunctional form by an electrophoretic mobility assay. The requirements for a separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are: (1) the folded form is sufficiently stable and (2) the samples are not heat-denatured before loading them onto the gel for the electrophoresis. Many folded and lipid-bilayer integrated β-TMPs may be extracted into micelles of SDS, but resist SDS-induced unfolding and the electrophoresis when SDS-PAGE is performed at room temperature or in a cold room. Unfolded β-TMPs bind SDS, which prevents their folding into β-barrel structure. These observations have been used to develop a simple assay to monitor the kinetics of β-barrel tertiary structure formation in a membrane environment by electrophoresis. A folding reaction of a β-TMP is initiated by dilution of the denaturant in the presence of preformed lipid bilayers, proteoliposomes, or membrane vesicles. After selected time intervals, samples are taken from the reaction and mixed with SDS containing Laemmli buffer to prevent further folding. The fraction of folded β-TMPs in each sample is analyzed by SDS-PAGE followed by densitometry of the gels. An advantage of this kinetic assay is that it not only allows a direct determination of fractions of folded and unfolded forms at a selected time during folding of the β-TMP into a membrane, but also facilitates the determination of the impact of folding factors, e.g., molecular chaoperones or folding machinery that most often have a different molecular mass and electrophoretic mobility. The assay has been very useful to examine how folding and insertion is affected by the structure of the phospholipids in the lipid bilayer and how folding machinery compensates for the presence of membrane lipids that retard folding and insertion of β-TMPs.
Purification of Bacterial Lipopolysaccharide, and How to Study Its Interactions with Proteins.
Saragliadis A, Hatlem D, Linke D
Methods Mol Biol · 2026 · PMID 42156675 · Publisher ↗
Lipopolysaccharides (LPS) are an important component of the outer membrane of Gram-negative bacteria. Although LPS is not homogeneous and it varies among bacterial species and even from strain to strain, there are signif...
Lipopolysaccharides (LPS) are an important component of the outer membrane of Gram-negative bacteria. Although LPS is not homogeneous and it varies among bacterial species and even from strain to strain, there are significantly conserved elements that confer and dictate the physicochemical properties of this molecule. Isolating and purifying LPS from its source organism provides the means to investigate the interactions of LPS molecules with other binding partners, such as proteins. Here, we describe methods to isolate LPS and discuss methods for interaction studies.
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