Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein-Lipid Interactions.

Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence-based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein-membrane interactions. Specifically, we discuss various environment-sensitive dyes, FRET probes, probes for short-distance measurements, and several probe-quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

Investigating membrane-binding properties of lipoxygenases using surface plasmon resonance.

Lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids and synthesize oxylipin products that drive important cellular signaling processes in plants and animals. While there has been indirect evidence presented for the interaction of mammalian LOXs with membranes, a quantitative study of the molecular details of LOX-membrane interactions is lacking. Here, we mimicked biological membranes using surface plasmon resonance (SPR) sensor chips derivatized with 2-D planar lipophilic anchors (2D LP) to capture liposomes of varying phospholipid compositions that self-assemble into lipid bilayers on the SPR chip. The sensor chip surfaces were then used to investigate the membrane-binding properties of model LOX enzymes. SPR binding assays displayed reproducible and stable liposome capture to the sensor chip surface that allowed for the detailed characterization of LOX-membrane interactions. Our studies demonstrate a calcium-dependence for the membrane binding activities of coral 8R-LOX and human 15-LOX-2. Furthermore, our data confirm the importance of key membrane insertion loop residues in each of these LOX enzymes for membrane binding activity. Experiments utilizing model plant and human LOXs reveal differences in membrane-binding specificities. Our study establishes and validates a robust SPR-based platform using 2D LP sensor chips that allows for the detailed study of LOX-membrane interactions under different experimental conditions, including altered membrane compositions. Collectively, this investigation improves our overall understanding of LOX-membrane interaction properties, and our SPR-based approach holds potential for future use in the development of LOX-based therapeutics.

Tunable lipid-coated nanoporous silver sheet for characterization of protein-membrane interactions by surface-enhanced Raman scattering (SERS).

Membrane environments affect protein structures and functions through protein-membrane interactions in a wide range of important biological processes. To better study the effects from the lipid's hydrophilic and hydrophobic interaction with protein on different membrane regions, we developed the lipid-coated nanoporous silver sheets to provide tunable supported lipid monolayer/bilayer environments for in situ surface-enhanced Raman vibrational spectroscopy (SERS) characterizations. Under the controllable surface pressure, lipid monolayer/bilayer was coated along the microscopic curved surface of nanoporous silver sheets to serve as a cell membrane mimic as well as a barrier to avoid protein denaturation while empowering the high SERS enhancements from the underlying metallic bases allowing detection sensitivity at low physiological concentrations. Moreover, we fine-tuned the lipid packing density and controlled the orientation of the deposited lipid bilayers and monolayers to directly monitor the protein structures upon interactions with various membrane parts/positions. Our results indicate that lysozyme adopted the α-helical structure in both hydrophilic and hydrophobic interaction with lipid membrane. Interestingly, alpha-synuclein folded into the α-helical structure on the negatively charged lipid heads, whereas the hydrophobic lipid tails induced the ß-sheet structural conversion of alpha-synuclein originated from its unstructured monomers. These direct observations on protein hydrophilic and hydrophobic interaction with lipid membrane might provide profound insights into the formation of the ß-sheet-containing alpha-synuclein oligomers for further membrane disruptions and amyloid genesis associated with Parkinson's disease. Hence, with the controllability and tunability of lipid environments, our platform holds great promise for more general applications in investigating the influences from membranes and the correlative structures of proteins under both hydrophilic and hydrophobic effects.

Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.

Insights into Membrane Curvature Sensing and Membrane Remodeling by Intrinsically Disordered Proteins and Protein Regions.

Cellular membranes are highly dynamic in shape. They can rapidly and precisely regulate their shape to perform various cellular functions. The protein's ability to sense membrane curvature is essential in various biological events such as cell signaling and membrane trafficking. As they are bound, these curvature-sensing proteins may also change the local membrane shape by one or more curvature driving mechanisms. Established curvature-sensing/driving mechanisms rely on proteins with specific structural features such as amphipathic helices and intrinsically curved shapes. However, the recent discovery and characterization of many proteins have shattered the protein structure-function paradigm, believing that the protein functions require a unique structural feature. Typically, such structure-independent functions are carried either entirely by intrinsically disordered proteins or hybrid proteins containing disordered regions and structured domains. It is becoming more apparent that disordered proteins and regions can be potent sensors/inducers of membrane curvatures. In this article, we outline the basic features of disordered proteins and regions, the motifs in such proteins that encode the function, membrane remodeling by disordered proteins and regions, and assays that may be employed to investigate curvature sensing and generation by ordered/disordered proteins.

Functional Delineation of a Protein-Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab-peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab-Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody-membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein-membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

Orientational Preferences of GPI-Anchored Ly6/uPAR Proteins.

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single ß-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal 'pre-orientation' of the LU domain for the receptor interaction.

Specific Protein-Membrane Interactions Promote Packaging of Metallo-ß-Lactamases into Outer Membrane Vesicles.

Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-ß-lactamases. The lipidated, membrane-anchored metallo-ß-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Here, we show that these interactions promote its packaging into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1 while being absent in VIM-2. These interactions correlate with an enhanced incorporation of IMP-1 compared to VIM-2 into OMVs. Disruption of these interactions in NDM-1 and IMP-1 impairs their inclusion into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that packaging of metallo-ß-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on specific molecular interactions.

Expanding MPEx Hydropathy Analysis to Account for Electrostatic Contributions to Protein Interactions with Anionic Membranes.

Hydropathy plots are a crucial tool to guide experimental design, as they generate predictions of protein-membrane interactions and their bilayer topology. The predictions are based on experimentally determined hydrophobicity scales, which provide an estimate for the propensity and stability of these interactions. A significant improvement to the accuracy of hydropathy analyses was provided by the development of the popular Wimley-White interfacial and octanol hydrophobicity scales. These scales have been previously incorporated into the freely available MPEx (Membrane Protein Explorer) online application. Here, we introduce a substantial update to MPEx that allows for the consideration of electrostatic contributions to the bilayer partitioning free energy. This component originates from the Coulombic attraction or repulsion of charges between proteins and membranes. Its inclusion in hydropathy calculations increases the accuracy of hydropathy plot predictions and extends their use to more complex systems (i.e., anionic membranes). We illustrate the application of this analysis to studies on the membrane selectivity of antimicrobial peptides, the membrane partitioning of ion-channel gating modifiers, and the amyloid proteins α-synuclein and Tau, as well as pH-dependent bilayer interactions of diphtheria toxin and apoptotic inhibitor Bcl-xL.

Modelling of the Citrus CCD4 Family Members: In Silico Analysis of Membrane Binding and Substrate Preference.

Coloring is one of the most important characteristics in commercial flowers and fruits, generally due to the accumulation of carotenoid pigments. Enzymes of the CCD4 family in citrus intervene in the generation of ß-citraurin, an apocarotenoid responsible for the reddish-orange color of mandarins. Citrus CCD4s enzymes could be capable of interacting with the thylakoid membrane inside chloroplasts. However, to date, this interaction has not been studied in detail. In this work, we present three new complete models of the CCD4 family members (CCD4a, CCD4b, and CCD4c), modeled with a lipid membrane. To identify the preference for substrates, typical carotenoids were inserted in the active site of the receptors and the protein-ligand interaction energy was evaluated. The results show a clear preference of CCD4s for xanthophylls over aliphatic carotenes. Our findings indicate the ability to penetrate the membrane and maintain a stable interaction through the N-terminal α-helical domain, spanning a contact surface of 2250 to 3250 Å2. The orientation and depth of penetration at the membrane surface suggest that CCD4s have the ability to extract carotenoids directly from the membrane through a tunnel consisting mainly of hydrophobic residues that extends up to the catalytic center of the enzyme.

Huntingtin: A Protein with a Peculiar Solvent Accessible Surface.

Taking advantage of the last cryogenic electron microscopy structure of human huntingtin, we explored with computational methods its physicochemical properties, focusing on the solvent accessible surface of the protein and highlighting a quite interesting mix of hydrophobic and hydrophilic patterns, with the prevalence of the latter ones. We then evaluated the probability of exposed residues to be in contact with other proteins, discovering that they tend to cluster in specific regions of the protein. We then found that the remaining portions of the protein surface can contain calcium-binding sites that we propose here as putative mediators for the protein to interact with membranes. Our findings are justified in relation to the present knowledge of huntingtin functional annotation.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL.

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL. The analysis reveals that the protonation state of the protein and cardiolipin content of the membrane modulate the orientation of the soluble head region (helices α1 through α7) and hence the exposure of its BH3-binding groove, which is required for its interaction with pro-apoptotic proteins.

K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function.

Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the ß2 and ß3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

NMR- and MD simulation-based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated.

The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and 15N relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe3049 and Trp3052) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules.

Membrane lipids are both the substrates and a mechanistically responsive environment of TMEM16 scramblase proteins.

Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo-EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function-related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer-tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs. © 2019 Wiley Periodicals, Inc.

The Implications for Cells of the Lipid Switches Driven by Protein-Membrane Interactions and the Development of Membrane Lipid Therapy.

The cell membrane contains a variety of receptors that interact with signaling molecules. However, agonist-receptor interactions not always activate a signaling cascade. Amphitropic membrane proteins are required for signal propagation upon ligand-induced receptor activation. These proteins localize to the plasma membrane or internal compartments; however, they are only activated by ligand-receptor complexes when both come into physical contact in membranes. These interactions enable signal propagation. Thus, signals may not propagate into the cell if peripheral proteins do not co-localize with receptors even in the presence of messengers. As the translocation of an amphitropic protein greatly depends on the membrane's lipid composition, regulation of the lipid bilayer emerges as a novel therapeutic strategy. Some of the signals controlled by proteins non-permanently bound to membranes produce dramatic changes in the cell's physiology. Indeed, changes in membrane lipids induce translocation of dozens of peripheral signaling proteins from or to the plasma membrane, which controls how cells behave. We called these changes "lipid switches", as they alter the cell's status (e.g., proliferation, differentiation, death, etc.) in response to the modulation of membrane lipids. Indeed, this discovery enables therapeutic interventions that modify the bilayer's lipids, an approach known as membrane-lipid therapy (MLT) or melitherapy.

Small GTPase peripheral binding to membranes: molecular determinants and supramolecular organization.

Small GTPases regulate many aspects of cell logistics by alternating between an inactive, GDP-bound form and an active, GTP-bound form. This nucleotide switch is coupled to a cytosol/membrane cycle, such that GTP-bound small GTPases carry out their functions at the periphery of endomembranes. A global understanding of the molecular determinants of the interaction of small GTPases with membranes and of the resulting supramolecular organization is beginning to emerge from studies of model systems. Recent studies highlighted that small GTPases establish multiple interactions with membranes involving their lipid anchor, their lipididated hypervariable region and elements in their GTPase domain, which combine to determine the strength, specificity and orientation of their association with lipids. Thereby, membrane association potentiates small GTPase interactions with GEFs, GAPs and effectors through colocalization and positional matching. Furthermore, it leads to small GTPase nanoclustering and to lipid demixing, which drives the assembly of molecular platforms in which proteins and lipids co-operate in producing high-fidelity signals through feedback and feedforward loops. Although still fragmentary, these observations point to an integrated model of signaling by membrane-attached small GTPases that involves a diversity of direct and indirect interactions, which can inspire new therapeutic strategies to block their activities in diseases.

Differing Membrane Interactions of Two Highly Similar Drug-Metabolizing Cytochrome P450 Isoforms: CYP 2C9 and CYP 2C19.

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.

Detecting Single-Molecule Dynamics on Lipid Membranes with Quenchers-in-a-Liposome FRET.

Tracking membrane-interacting molecules and visualizing their conformational dynamics are key to understanding their functions. It is, however, challenging to accurately probe the positions of a molecule relative to a membrane. Herein, a single-molecule method, termed LipoFRET, is reported to assess interplay between molecules and liposomes. It takes advantage of FRET between a single fluorophore attached to a biomolecule and many quenchers in a liposome. This method was used to characterize interactions between α-synuclein (α-syn) and membranes. These results revealed that the N-terminus of α-syn inserts into the membrane and spontaneously transitions between different depths. In contrast, the C-terminal tail of α-syn is regulated by calcium ions and floats in solution in two conformations. LipoFRET is a powerful tool to investigate membrane-interacting biomolecules with sub-nanometer precision at the single-molecule level.

Membrane pore formation at protein-lipid interfaces.

Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aß1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further.