Single enzyme studies reveal the existence of discrete functional states for monomeric enzymes and how they are "selected" upon allosteric regulation.

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme's major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones.

Intermembrane docking reactions are regulated by membrane curvature.

The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle-vesicle pairs of different diameter (30-200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30-60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2-10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes.

A structural analysis of M protein in coronavirus assembly and morphology.

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

How curved membranes recruit amphipathic helices and protein anchoring motifs.

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte.

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations in membrane thickness. We measured membrane thickness directly from electron micrographs of sections of fixed mucosal tissue. Indeed, mapping of the microvillar membrane revealed a biphasic distribution of membrane thickness. As a point of reference the thickness distribution of the basolateral membrane was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm(2). In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold labeling of alkaline phosphatase, a well documented raft marker. Strikingly, the alkaline phosphatase localized to distinct regions of the membrane in a pattern similar to the observed distribution of DITs. Although we were unable to measure membrane thickness directly on the immunogold labeled specimens, and thereby establish an unequivocal connection between DITs and rafts, we conclude that the brush border membrane of the enterocyte contains microdomains distinguishable both by membrane morphology and protein composition.

A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles.

We report a novel approach to quantitatively determine complete size distributions of surface-bound objects using fluorescence microscopy. We measure the integrated intensity of single particles and relate it to their size by taking into account the object geometry and the illumination profile of the microscope, here a confocal laser scanning microscope. Polydisperse (as well as monodisperse) size distributions containing objects both below and above the optical resolution of the microscope are recorded and analyzed. The data is collected online within minutes, which allows the user to correlate the size of an object with the response from any given fluorescence-based biochemical assay. We measured the mean diameter of extruded fluorescently labeled lipid vesicles using the proposed method, dynamic light scattering, and cryogenic transmission electron microscopy. The three techniques were in excellent agreement, measuring the same values within 7-9%. Furthermore we demonstrated here, for the first time that we know of, the ability to determine the full size distribution of polydisperse samples of nonextruded lipid vesicles. Knowledge of the vesicle size distribution before and after extrusion allowed us to propose an empirical model to account for the effect of extrusion on the complete size distribution of vesicle samples.

Micro-droplet arrays for micro-compartmentalization using an air/water interface.

Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aß42 peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.

Constructing size distributions of liposomes from single-object fluorescence measurements.

We describe in detail a simple technique to construct the size distribution of liposome formulations from single-object fluorescence measurements. Liposomes that are fluorescently labeled in their membrane are first immobilized on a surface at dilute densities and then imaged individually using epi-fluorescence microscopy. The integrated intensities of several thousand single liposomes are collected and evaluated within minutes by automated image processing, using the user-friendly freeware ImageJ. The mean intensity of the liposome population is then calculated and scaled in units of length (nm) by relating the intensity data to the mean diameter obtained from a reference measurement with dynamic light scattering. We explain the process of constructing the size distributions in a step-by-step manner, starting with the preparation of liposomes through the final acquisition of size histograms. Detailed advice is given concerning critical parameters of image acquisition and processing. Size histograms constructed from single-particle measurements provide detailed information on complex distributions that may be easily averaged out in ensemble measurements (e.g., light scattering). In addition, the technique allows accurate measurements of polydisperse samples (e.g., nonextruded liposome preparations).