Real-time binding kinetics of ligand-receptor interactions

In principle, a major goal in drug development is to design a high-affinity compound with an appropriate pharmacological response, which requires understanding of physical processes underlying the drug-target interaction, affinity and specificity. This drug key information can be obtained by measuring drug binding kinetics in real time as the quantity of a ligand binding to a receptor immobilized on a biosensor chip surface. Almost all drugs independently of their targets interact with a complex cell membrane that incorporate various transmembrane proteins whose impact cannot be disregarded. Therefore, it is highly desirable that preclinical drug development is performed under conditions that are as close as possible to a native cell environment. The above reasons enhance the need for alternative approaches, such as whole-cell assays that are able to examine in vitro potential drug compounds. However, nowadays fragments of cell membranes have to be routinely isolated from the native cell environment due to limitations of currently available label-free technologies. In recent years, attempts were made to measure binding kinetics on living bacteria by surface plasmon resonance-based biosensors. Nevertheless, the experiments were not fully successful because of an insufficient penetration depth of the sensing evanescent field into living bacteria. Label-free biosensors based on metal-free photonic crystals support electromagnetic surface waves with a deeper penetration depth into the sample medium offering a solution for measuring binding interactions with living bacterial and, possibly, also eukaryotic cells. The deeper penetration of the surface waves creates a significant advantage for this non-disruptive technology over conventional immunoassays such as ELISA. For these reasons, label-free biosensors have been attracting a growing interest from pharmaceutical companies and research institutions by avoiding any experimental artefacts induced by labeling and allowing for real-time kinetic measurements. In this work, I contributed to the development of a label-free optical biosensor based on a photonic crystal that can be exploited for living bacteria. Moreover, I designed an experimental protocol and demonstrated how binding kinetics of various ligands, including antibodies and bacteriophages can be measured by the biosensor in vitro. Furthermore, this study attempts to apply the biosensor technology for bacterial viability tests exploring the ways in which the biosensor can detect metabolic activity of bacteria. Additionally, we performed theoretical studies on application of the Hillâs and Mathieuâs equations to study the effect of the photonic crystal design on the wave equation describing electromagnetic field distribution in the photonic crystal. This Thesis work demonstrates capabilities of a versatile biophysical method based on light interaction with living matter. In particular, the device sensitivity allows for measurements on living intact cells in vitro, which is a key technological advantage. Finally, this Thesis provides experimental evidence for future applications of the photonic crystal-based biosensor for antibody and bacteriophage therapies contributing to implementation of this technology in drug development process.

Kinetics of Antibody Binding to Membranes of Living Bacteria Measured by a Photonic Crystal-Based Biosensor

Carine Ben Adiba, Giovanni Dietler, Ekaterina Rostova

Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s-and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to K-D = 6.2 +/- 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.

Mdpi Ag2016

Assembly Mechanisms and Cellular Effects of Bacterial Pore-Forming Toxins

Mirko Bischofberger

The correct assembly of macromolecular protein complexes such as ribosomes or multisubunit membrane channels is essential for their function. Alterations in the process can lead to disease either by loss of function or by acquisition of toxic function. However, the precise mechanisms by which multimeric protein complexes assemble are generally poorly understood owing to the difficulty in reconstituting the process in-vitro. Thus, systems that undergo dynamic assembly and disassembly in solution, such as cytoskeletal proteins, have been particularly attractive. By contrast, the assembly of multi-subunit membrane protein complexes has been notably difficult to study. An interesting, somewhat in between, situation is provided by pore-forming proteins, the best-characterized subclass of which are bacterial pore-forming toxins (PFTs). These proteins can generally be produced recombinantly and in a highly stable soluble monomeric form, but assemble into homo-oligomeric ring-like structures upon addition to cells expressing the appropriate cell surface receptor. These rings concomitantly, or subsequently depending of on the PFT, undergo a conformational change that leads to exposure of hydrophobic surfaces and spontaneous membrane insertion. The built pores then perforate the membrane of the host cell. PFTs do not only occur in bacteria and have a very broad taxonomic distribution, occurring in organisms such as parasites, plants and mammals. However, for pathogenic organisms the PFTs are major virulence factors contributing to the disease caused by the organisms producing them. For this reason bacterial PFTs have been studied extensively during the last century and a lot of insight into their mode of action has been gained. However, despite the increasing knowledge on PFTs, the mechanisms and the kinetics of self-assembly of these complexes remain largely enigmatic. In particular it is unknown whether oligomerization occurs through the sequential addition of monomers or through interaction of multimeric intermediates, whether oligomerization is the rate-limiting step during the pore-formation process and whether the same rules apply to all toxins. This is mostly due to the fact that intermediates have not been visualized by either biochemical or structural methods. Also, with few exceptions, functional assays on the activity of PFTs have always been performed at the level of a population of cells. The aim of this thesis was to measure pore-formation at the single cell level to extract mechanistic information from the analysis of the stochasticity of the pore-formation process. We chose to study three PFTs produced by human pathogens: aerolysin, PFO and Cytolysin A (ClyA). Aerolysin is produced by Aeromonas hydrophila and forms pores of 1-2 nm in diameter, perfringolysin O (PFO) occurs in Clostridium perfringensis and is a cholesterol-dependent cytolysin (CDC) that builds channels of 25-30 nm. Both of these PFTs build structurally similar pores made of transmembrane β-barrels even though the number of strands in the barrel varies by more than 1 order of magnitude. On the contrary, ClyA is a PFT produced by both E. coli and Salmonella enterica strains and forms rings of 12-13 protomers with the membrane spanning domain composed of a-helices. Hence, the pores formed by these three PFTs vary greatly from one another and are representative for the diversity of PFTs. We measured cell permeabilisation at the single cell level using two independent live-cell imaging methods, in erythrocytes and in nucleated cells. Both assays revealed that the assembly mechanism of PFTs is stochastic in its nature and that lowering the initial monomer concentration increases the variability and average time to successfully build first functional pores. The measured stochasticity reflects the chemical kinetics behind the assembly reaction and therefore carries important information about the underlying mechanism. In fact, analysis of the stochasticity of the processes led to the robust determination for each toxin of the minimal number of independent rate-limiting steps required for pore formation. Furthermore, scaling relationships in our data upon changes in initial toxin concentration constrain the possible underlying mechanisms for assembly and reveal that the steps leading to the aerolysin heptamer are composed of at least seven independent reactions whose rates scale with toxin concentration. Taken together, our findings suggest that that what is limiting for aerolysin is the conversion for each monomer to an assembly competent state, while for PFO a single limiting step is observed, likely corresponding to membrane insertion. A second aim of this thesis was to clarify the cellular consequences that occur upon pore formation by different PFTs and to find out how PFT-affected cells sense this event. This was done in collaboration with other members of the lab. Our results suggest that the cells monitor the breaches in the plasma membrane by assessing the correct homeostasis of the potassium ion inside the cell through a yet unknown mechanism. Interestingly, the cells seem to "monitor" the integrity of the membrane through the most upstream effect possible after pore formation, namely the flux of ions. Incidentally, the assays we developed in the first part of this thesis to "monitor" initial pore formation events are based on the same principle, namely the immediate loss of gradients that occur upon pore formation.

EPFL2011

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.