Melittin-Phospholipase A2 Synergism Is Mediated by Liquid-Liquid Miscibility Phase Transition in Giant Unilamellar Vesicles.

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations.

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A2 (PLA2), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA2 activity is 25-fold higher. Tracking of single-molecule PLA2 revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA2 work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.

EphrinB2 clustering by Nipah virus G is required to activate and trap F intermediates at supported lipid bilayer-cell interfaces.

Paramyxovirus membrane fusion requires an attachment protein that binds to a host cell receptor and a fusion protein that merges the viral and host membranes. For Nipah virus (NiV), the G attachment protein binds ephrinB2/B3 receptors and activates F-mediated fusion. To visualize dynamic events of these proteins at the membrane interface, we reconstituted NiV fusion activation by overlaying F- and G-expressing cells onto ephrinB2-functionalized supported lipid bilayers and used TIRF microscopy to follow F, G, and ephrinB2. We found that G and ephrinB2 form clusters and that oligomerization of ephrinB2 is necessary for F activation. Single-molecule tracking of F particles revealed accumulation of an immobilized intermediate upon activation. We found no evidence for stable F-G protein complexes before or after activation. These observations lead to a revised model for NiV fusion activation and provide a foundation for investigating other multicomponent viral fusion systems.

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces.

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions-the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton's tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions. [BMB Reports 2021; 54(3): 157-163].

Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions.

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.

Switch-like activation of Bruton's tyrosine kinase by membrane-mediated dimerization.

The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on protein-ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton's tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a phosphatidylinositol (3-5)-trisphosphate (PIP3) sensing mechanism that achieves switch-like sensitivity to PIP3 levels, surpassing the intrinsic affinity discrimination of PIP3:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Studies in live cells confirm that mutations at the dimer interface and peripheral site produce effects comparable to that of the kinase-dead Btk in vivo. These results demonstrate how a single protein module can institute an allosteric counting mechanism to achieve high-precision discrimination of ligand concentration. Furthermore, this activation mechanism distinguishes Btk from other Tec family member kinases, Tec and Itk, which we show are not capable of dimerization through their PH-TH modules. This suggests that Btk plays a critical role in the stringency of the B cell response, whereas T cells rely on other mechanisms to achieve stringency.

A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS.

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-molecule assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS molecules on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (~50 seconds) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)-Grb2-SOS phosphotyrosine-driven phase transition at the membrane.

Spatiomechanical Modulation of EphB4-Ephrin-B2 Signaling in Neural Stem Cell Differentiation.

Interactions between EphB4 receptor tyrosine kinases and their membrane-bound ephrin-B2 ligands on apposed cells play a regulatory role in neural stem cell differentiation. With both receptor and ligand constrained to move within the membranes of their respective cells, this signaling system inevitably experiences spatial confinement and mechanical forces in conjunction with receptor-ligand binding. In this study, we reconstitute the EphB4-ephrin-B2 juxtacrine signaling geometry using a supported-lipid-bilayer system presenting laterally mobile and monomeric ephrin-B2 ligands to live neural stem cells. This experimental platform successfully reconstitutes EphB4-ephrin-B2 binding, lateral clustering, downstream signaling activation, and neuronal differentiation, all in a configuration that preserves the spatiomechanical aspects of the natural juxtacrine signaling geometry. Additionally, the supported bilayer system allows control of lateral movement and clustering of the receptor-ligand complexes through patterns of physical barriers to lateral diffusion fabricated onto the underlying substrate. The results from this study reveal a distinct spatiomechanical effect on the ability of EphB4-ephrin-B2 signaling to induce neuronal differentiation. These observations parallel similar studies of the EphA2-ephrin-A1 system in a very different biological context, suggesting that such spatiomechanical regulation may be a common feature of Eph-ephrin signaling.

K-Ras4B Remains Monomeric on Membranes over a Wide Range of Surface Densities and Lipid Compositions.

Ras is a membrane-anchored signaling protein that serves as a hub for many signaling pathways and also plays a prominent role in cancer. The intrinsic behavior of Ras on the membrane has captivated the biophysics community in recent years, especially the possibility that it may form dimers. In this article, we describe results from a comprehensive series of experiments using fluorescence correlation spectroscopy and single-molecule tracking to probe the possible dimerization of natively expressed and fully processed K-Ras4B in supported lipid bilayer membranes. Key to these studies is the fact that K-Ras4B has its native membrane anchor, including both the farnesylation and methylation of the terminal cysteine, enabling detailed exploration of possible effects of cholesterol and lipid composition on K-Ras4B membrane organization. The results from all conditions studied indicate that full-length K-Ras4B lacks intrinsic dimerization capability. This suggests that any lateral organization of Ras in living cell membranes likely stems from interactions with other factors.

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Graphene-Templated Supported Lipid Bilayer Nanochannels.

The use of patterned substrates to impose geometrical restriction on the lateral mobility of molecules in supported lipid membranes has found widespread utility in studies of cell membranes. Here, we template-pattern supported lipid membranes with nanopatterned graphene. We utilize focused ion beam milling to pattern graphene on its growth substrate, then transfer the patterned graphene to fresh glass substrates for subsequent supported membrane formation. We observe that graphene functions as an excellent lateral diffusion barrier for supported lipid bilayers. Additionally, the observed diffusion dynamics of lipids in nanoscale graphene channels reveal extremely low boundary effects, a common problem with other materials. We suggest this is attributable to the ultimate thinness of graphene.

Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation.

Ras, a small GTPase found primarily on the inner leaflet of the plasma membrane, is an important signaling node and an attractive target for anticancer therapies. Lateral organization of Ras on cellular membranes has long been a subject of intense research; in particular, whether it forms dimers on membranes as part of its regulatory function has been a point of great interest. Here we report Ras dimer formation on membranes by Type II photosensitization reactions, in which molecular oxygen mediates the radicalization of proteins under typical fluorescence experimental conditions. The presence of Ras dimers on membranes was detected by diffusion-based fluorescence techniques including fluorescence correlation spectroscopy and single particle tracking, and molecular weights of the stable covalently coupled species were confirmed by gel electrophoresis. Fluorescence spectroscopy implicates interprotein dityrosine as one of the dimerization motifs. The specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction, and point mutations affecting surface tyrosines are observed to alter dimerization potential. The photosensitization reactions are reflective of physiological oxidative stress induced by reactive oxygen species, suggesting such processes may occur naturally and influence signaling pathways in cells.

Fast dynamics of HP35 for folded and urea-unfolded conditions.

The changes in fast dynamics of HP35 with a double CN vibrational dynamics label (HP35-P(2)) as a function of the extent of denaturation by urea were investigated with two-dimensional infrared (2D IR) vibrational echo spectroscopy. Cyanophenylalanine (PheCN) replaces the native phenylalanine at two residues in the hydrophobic core of HP35, providing vibrational probes. NMR data show that HP35-P(2) maintains the native folded structure similar to wild type and that both PheCN residues share essentially the same environment within the peptide. A series of time-dependent 2D IR vibrational echo spectra were obtained for the folded peptide and the increasingly unfolded peptide. Analysis of the time dependence of the 2D spectra yields the system's spectral diffusion, which is caused by the sampling of accessible structures of the peptide under thermal equilibrium conditions. The structural dynamics become faster as the degree of unfolding is increased.

Conformational dynamics and stability of HP35 studied with 2D IR vibrational echoes.

Two-dimensional infrared (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core were used as a vibrational probe. Experiments were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P(2)) within the wild-type sequence, as well as on HP-35 containing a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an additional CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR experiments reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle's increased stability.

Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand.

Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.

Influence of histidine tag attachment on picosecond protein dynamics.

Polyhistidine affinity tags are routinely employed as a convenient means of purifying recombinantly expressed proteins. A tacit assumption is commonly made that His tags have little influence on protein structure and function. Attachment of a His tag to the N-terminus of the robust globular protein myoglobin leads to only minor changes to the electrostatic environment of the heme pocket, as evinced by the nearly unchanged Fourier transform infrared spectrum of CO bound to the heme of His-tagged myoglobin. Experiments employing two-dimensional infrared vibrational echo spectroscopy of the heme-bound CO, however, find that significant changes occur to the short time scale (picoseconds) dynamics of myoglobin as a result of His tag incorporation. The His tag mainly reduces the dynamics on the 1.4 ps time scale and also alters protein motions of myoglobin on the slower, >100 ps time scale, as demonstrated by the His tag's influence on the fluctuations of the CO vibrational frequency, which reports on protein structural dynamics. The results suggest that affinity tags may have effects on protein function and indicate that investigators of affinity-tagged proteins should take this into consideration when investigating the dynamics and other properties of such proteins.

Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments.

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy.

A series of two-dimensional infrared vibrational echo experiments performed on nitrile-labeled villin headpiece [HP35-(CN)(2)] is described. HP35 is a small peptide composed of three alpha helices in the folded configuration. The dynamics of the folded HP35-(CN)(2) are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held constant by adding glycerol. For the folded peptide, the addition of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity constant as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent molecules, in contrast to the observations on the folded peptide.