Single-Particle Tracking of Thermomyces lanuginosus Lipase Reveals How Mutations in the Lid Region Remodel Its Diffusion.

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water-lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases' function is important for designing improved variants. Lipases' function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes' diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent.

Membrane anchoring facilitates colocalization of enzymes in plant cytochrome P450 redox systems.

Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes.

Single-Molecule Study of Thermomyces lanuginosus Lipase in a Detergency Application System Reveals Diffusion Pattern Remodeling by Surfactants and Calcium.

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

Direct observation of Thermomyces lanuginosus lipase diffusional states by Single Particle Tracking and their remodeling by mutations and inhibition.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Stochastic geometry sensing and polarization in a lipid kinase-phosphatase competitive reaction.

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.

Single Proteoliposome High-Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs.

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (ß2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.

One-way membrane trafficking of SOS in receptor-triggered Ras activation.

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

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.

Direct observation of proton pumping by a eukaryotic P-type ATPase.

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Monitoring the Waiting Time Sequence of Single Ras GTPase Activation Events Using Liposome Functionalized Zero-Mode Waveguides.

Activation of small GTPases of the Ras superfamily by guanine nucleotide exchange factors (GEFs) is a key step in numerous cell signaling processes. Unveiling the detailed molecular mechanisms of GEF-GTPase signaling interactions is of great importance due to their central roles in cell biology, including critical disease states, and their potential as therapeutic targets. Here we present an assay to monitor individual Ras activation events catalyzed by single molecules of the GEF Son of Sevenless (SOS) in the natural membrane environment. The assay employs zero-mode waveguide (ZMW) nanostructures containing a single Ras-functionalized liposome. The ZMWs facilitate highly localized excitation of fluorophores in the vicinity of the liposome membrane, allowing direct observation of individual Ras activation events as single SOS enzymes catalyze exchange of unlabeled nucleotides bound to Ras with fluorescently labeled nucleotides from solution. The system is compatible with continuous recording of long sequences of individual enzymatic turnover events over hour-long time scales. The single turnover waiting time sequence is a molecular footprint that details the temporal characteristics of the system. Data reported here reveal long-lived activity states that correspond to well-defined conformers of SOS at the membrane. Liposome functionalized ZMWs allow for studies of nucleotide exchange reactions at single GTPase resolution, providing a platform to gauge the mechanisms of these processes.

Interferometric Detection of Single Gold Nanoparticles Calibrated against TEM Size Distributions.

Single nanoparticle analysis: An interferometric optical approach calibrates sizes of gold nanoparticles (AuNPs) from the interference intensities by calibrating their interferometric signals against the corresponding transmission electron microscopy measurements. This method is used to investigate whether size affects the diffusion behavior of AuNPs conjugated to supported lipid bilayer membranes and to multiplex the simultaneous detection of three different AuNP labels.

Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes.

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the ß2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

H-Ras forms dimers on membrane surfaces via a protein-protein interface.

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to be ∼1 × 10(3) molecules/µm(2), and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.

Activation-triggered subunit exchange between CaMKII holoenzymes facilitates the spread of kinase activity.

The activation of the dodecameric Ca(2+)/calmodulin dependent kinase II (CaMKII) holoenzyme is critical for memory formation. We now report that CaMKII has a remarkable property, which is that activation of the holoenzyme triggers the exchange of subunits between holoenzymes, including unactivated ones, enabling the calcium-independent phosphorylation of new subunits. We show, using a single-molecule TIRF microscopy technique, that the exchange process is triggered by the activation of CaMKII, and that exchange is modulated by phosphorylation of two residues in the calmodulin-binding segment, Thr 305 and Thr 306. Based on these results, and on the analysis of molecular dynamics simulations, we suggest that the phosphorylated regulatory segment of CaMKII interacts with the central hub of the holoenzyme and weakens its integrity, thereby promoting exchange. Our results have implications for an earlier idea that subunit exchange in CaMKII may have relevance for information storage resulting from brief coincident stimuli during neuronal signaling. DOI: http://dx.doi.org/10.7554/eLife.01610.001.

Geometrical membrane curvature as an allosteric regulator of membrane protein structure and function.

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane ß-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ~1/40 nm(-1)) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (~40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ~50 mN/m and a curvature-imposed protein deformation energy of ~7 kBT. Such substantial energies have been shown to conformationally activate, or unfold, ß-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.

Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience.

One of the major bottlenecks in the development of biochips is maintaining the structure and function of biomolecules when interfacing them with hard matter (glass, plastics, metals, etc.), a challenge that is exacerbated during miniaturization that inevitably increases the interface to volume ratio of these devices. Biochips based on immobilized vesicles circumvent this problem by encapsulating biomolecules in the protective environment of a lipid bilayer, thus minimizing interactions with hard surfaces. Here we review the development of biochips based on arrays of single nanoscale vesicles, their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high throughput, single vesicle biochips are still a niche technology that has yet to establish its commercial relevance.

Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics.

Handling and mixing ultrasmall volumes of reactants in parallel can increase the throughput and complexity of screening assays while simultaneously reducing reagent consumption. Microfabricated silicon and plastic can provide reliable fluidic devices, but cannot typically handle total volumes smaller than ∼1 × 10(-12) l. Self-assembled soft matter nanocontainers can in principle significantly improve miniaturization and biocompatibility, but exploiting their full potential is a challenge due to their small dimensions. Here, we show that small unilamellar lipid vesicles can be used to mix volumes as small as 1 × 10(-19) l in a reproducible and highly parallelized fashion. The self-enclosed nanoreactors are functionalized with lipids of opposite charge to achieve reliable fusion. Single vesicles encapsulating one set of reactants are immobilized on a glass surface and then fused with diffusing vesicles of opposite charge that carry a complementary set of reactants. We find that ∼85% of the ∼1 × 10(6) cm(-2) surface-tethered nanoreactors undergo non-deterministic fusion, which is leakage-free in all cases, and the system allows up to three to four consecutive mixing events per nanoreactor.

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity.

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca(2+) dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca(2+) accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca(2+) accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) ∼ 1 min and t(mix) ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.