An Integrated Method for Profiling Lipid–Protein Interactions Using Multifunctional Lipid Probes

Statistical mechanics of lipid membranes. Protein correlation functions and lipid ordering

The study of lipid-protein interactions is crucial for understanding reactions of proteins involved in lipid metabolism, lipid transport, and lipid signaling. Different detection methods can be employed for the identification of lipid-binding interactions. Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) spectroscopy enable real-time monitoring of lipid protein interactions and provide thermodynamic parameters of the interacting partners. However, these technologies depend on the availability of the large equipment, limiting the practicability in many laboratories. Protein-lipid overlay assays are a simple first approach to screen for protein interactions with different lipids or lipid intermediates and are independent of large equipment. Subsequently, specific interactions can be analyzed in detail using protein-liposome association assays.

Lipid–protein interactions are central to maintaining the structural and functional balance of biological membranes, influencing a wide array of cellular processes. These interactions, however, become pathological in neurodegenerative diseases (NDDs), such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. In these disorders, the misfolding and aggregation of proteins like amyloid-beta (Aβ), alphasynuclein (α-syn), and mutant huntingtin (mHTT) disrupt the lipid bilayer, compromising membrane integrity, fluidity, and signaling. In this review we explore the critical role of lipid–protein interactions in NDDs, emphasizing how protein misfolding leads to toxic aggregates that embed into membranes, triggering neurotoxic events. Advanced spectroscopic techniques have been instrumental in studying these molecular interactions. Photon-based methods, including Förster resonance energy transfer (FRET), circular dichroism (CD), and Raman spectroscopy, provide real-time insights into protein aggregation and lipid membrane dynamics. Neutron-based techniques, such as neutron reflectometry and small-angle neutron scattering (SANS), further enhance the resolution of lipid–protein interactions, particularly in the context of neurodegenerative aggregation. Moreover, the review highlights the significance of lipid microdomains, particularly cholesterol-rich lipid rafts, which act as platforms for protein aggregation, influencing disease progression. Therapeutic strategies aimed at targeting these lipid–protein interfaces are also discussed, with a focus on how spectroscopic insights have driven the development of drugs that stabilize membrane integrity or prevent toxic aggregation. Finally, the integration of spectroscopy with computational models, such as molecular dynamics (MD) simulations, is proposed as a promising approach to further unravel the complex dynamics of lipid–protein interactions, providing a more complete picture of disease mechanisms.

Specific interactions of protein side chains and lipid membranes regulate the localization, orientation, and activity of many peripheral proteins. Here, we introduce a modification of the coarse-grained MARTINI protein model, called 'side chain fix' (scFix), that was necessary and sufficient to correctly sample the side chain dynamics of β-strands in several globular proteins. When compared to μs long atomistic simulations or previous experimental findings, scFix MARTINI simulations reproduced all key interactions between the well-studied PLC-δ1 pleckstrin homology domain and a phosphatidylinositol-4,5-bisphosphate (PIP2) containing lipid membrane. Moreover, the extended runtime and higher sampling speed enabled the systematic mapping of the protein's rolling motion at the membrane, the identification of short-lived and stable binding orientations, as well as the verification and prediction of already known and of novel transient PIP2 binding sites. scFix also showed promise to maintain proper side chain orientation in other secondary structural motifs of the α-spectrin SH3 domain, the B1 domain of protein G, and the villin headpiece. This suggests that scFix improves on the predictive power of MARTINI simulations regarding protein-lipid and protein-ligand interactions.

Lipid rafts are specialized microdomains of the plasma membrane that interact with proteins involved in vesicular transport and signaling mechanisms. Lipid rafts are enriched in cholesterol, sphingolipids, and scaffolding proteins, such as caveolins and septins.1–5 Although the precise functions of lipid rafts are still controversial and largely based on in vitro studies there is evidence that lipid rafts and associated scaffolding proteins may be involved in the pathogenesis of neurologic disease. For example, mutations of caveolin-3 have been associated with phenotypically diverse muscle disorders.6 Lipid rafts may also be the site of abnormal processing of amyloid precursor protein and prion protein.7 Impaired septin 4 function may contribute to synuclein accumulation and neurotoxicity in Parkinson disease (PD).8–10 There are several comprehensive reviews on the composition and potential functions of lipid rafts and their associated scaffold proteins.1–5,10 This brief review selectively focuses on some of these concepts that may have implications for neurologic disease. The plasma membrane is a lipid bilayer organized into discrete compartments or microdomains through lipid-lipid, lipid-protein, and membrane-cytoskeletal interactions.1,2 Lipids rafts are microdomains that are particularly rich in cholesterol and sphingolipids (glycolipids and sphingomyelin) and act as platforms for colocalization and interaction of proteins involved in vesicular transport, signaling mechanisms, and other functions2–4 (table). Proteins associated with lipid rafts include cell surface proteins that are attached to the membrane by a glycosyl phosphatidylinositol (GPI) anchor and proteins are that covalently modified by conjugation with saturated fatty acids, such as palmitic acid, present in the sphingolipids side chain. Lipid rafts are also characterized by their interactions with the cytoskeleton.1,2 Two types of lipid rafts are caveolae (little caves, figure) and planar lipid rafts.4 View this table: Table Some of the proteins associated with lipid rafts and potential functional implications Figure Examples of possible …

The use of light polarization properties in the analysis of fluorescence images has driven a large amount of research toward the measurement of orientational behavior of molecules in cells, in particular in their membranes. This field has been recently revisited to enlarge the possibilities of polarization-resolved fluorescence microscopy. We show that this technique allows retrieving a wealth of information on the constraints that hinder rotational mobility of lipid probes and proteins in membranes, bringing thus new insights on inter-proteins and lipid-protein interactions, on membrane morphology at the sub-diffraction length scale and on local membrane physical properties such as viscosity.

Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function. Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain. We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting. We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures. However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups. Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecule's C-terminus. One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane. The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes. Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting.

Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of the H+-ATPase and the Na+/K+-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca2+ signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid – protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na+/K+-ATPase, and other P-type ATPases. Keywords: Ca2+-ATPase, hydrophobic photo-labeling, lipid-protein interaction, membrane protein, Na+/K+-ATPase, PMCA, P-type ATPase, photo-labeling group TPD, 8-(5'-azido-O-hexanoylsalicylami-do)octanoic acid (AS86), Ca2+-ATPase activity, SERCA

Denervation alters protein–lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.)

Lipids play importantmodulatory and structural roles for membraneproteins. Molecular dynamics simulations are frequently used to provideinsights into the nature of these protein–lipid interactions.Systematic comparative analysis requires tools that provide algorithmsfor objective assessment of such interactions. We introduce PyLipID,a Python package for the identification and characterization of specificlipid interactions and binding sites on membrane proteins from moleculardynamics simulations. PyLipID uses a community analysis approach forbinding site detection, calculating lipid residence times for boththe individual protein residues and the detected binding sites. Toassist structural analysis, PyLipID produces representative boundlipid poses from simulation data, using a density-based scoring function.To estimate residue contacts robustly, PyLipID uses a dual-cutoffscheme to differentiate between lipid conformational rearrangementswhile bound from full dissociation events. In addition to the characterizationof protein–lipid interactions, PyLipID is applicable to analysisof the interactions of membrane proteins with other ligands. By combiningautomated analysis, efficient algorithms, and open-source distribution,PyLipID facilitates the systematic analysis of lipid interactionsfrom large simulation data sets of multiple species of membrane proteins.

Protein–lipid interactions in membrane trafficking at the Golgi complex

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain

The purified Na+,Mg2(+)-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid-crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid-crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.

Functional lipidomics: the roles of specialized lipids and lipid–protein interactions in modulating neuronal function

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (Fv) and the maximal (Fm) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the α-helix content (1656 cm-1), a parallel increase in the disordered segments (1651 cm-1), a decrease in the intramolecular β-sheet (1636 cm-1) content and the concomitant appearance of an intermolecular β-structure (1621 cm-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular β-structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these ‘lipidated’ proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.