Labelled Ligand Binding Assays Receptors, possess an extracellular domain for binding ligands , a transmembrane domain, and an intracellular or cytoplasmic domain. The first step is the binding of ligands to receptors at the cell surface. Bound receptors can activate various intracellular enzymes and entire cascades of intracellular reactions. Some of these regions trigger short-term (of the order of milliseconds to minutes) responses. Long-term responses, such as those requiring protein synthesis, involve additional molecular interactions. Each step is characterized by parameters such as rate constants and concentrations which are in principle measurable and alterable.

Principles of receptor binding assays There are several stages in the development of a binding program: initial choices, establishment of assay conditions, validation, application to novel ligands and quantitative analysis of the resulting data to derive binding parameters for the ligands

Three types of receptor binding experiments may be performed Kinetic experiments Saturation experiments Competition/modulation experiments

Approaches for distinguishing cell surface binding events from subsequent receptor trafficking processes (1) performing experiments at lowered temperatures, typically 4-15 °C (2) use of pharmacologic agents (3) use of isolated membranes possessing receptors (4) development of experimental protocols in which particular steps can be isolated by judicious choice of time scales or measurement procedure (5) estimation of all rate constants together using a complete model and comprehensive set of kinetic experiments involving a wider range of measurements physiological relevance !! Extrinsic variables

Receptor Choices Whole tissue extract Whole cells Cell membrane preparations

Radioactively labeled ligands Radioactivity is measured in Curies (Ci) or in Becquerels scintillation counters 3H, 14C, 35S, 32P, 125I Scintillant converts the radiation into light quanta that could be registered as counts by a photomultiplier. Choice of label is important Separation step is needed: vaccum filtration, time of separation!

Fluorescently-labeled ligands promising alternatives to radioactive assays safe to use, bright and stable probes have been developed over the past two decades and are now commercially available potent fluorescent ligands have been designed and synthesized for numerous receptors. instruments needed to measure fluorescence are also becoming more sensitive With spectrofluorimetric approaches, bound and free ligand can be distinguished However, It adds steric hindrance as well as hydrophobicity modifications

Direct Measurement of Fluorescent Intensity of Ligand Bound to receptors measurement of the fluorescent ligand bound fraction after separation from the free fraction auto-fluorescence of biological preparations non-specific binding of the fluorescent ligand the hydrophobicity of the fluorophore linked to the ligand can increase its non-specific binding

Fluorescence Polarization Assays measure more precisely the amount of bound fluorescent ligand. Based on the excitation of the biological sample with polarized light. Constrained fluorophores (bound) will emit highly polarized fluorescence. On the other hand, freely moving fluorophores which can easily rotate during the short period between their excitation and the emission of fluorescence will scramble this polarization.

FRET-Based Binding Assays FRET occurs between a ‘donor’ fluorophore and an ‘acceptor’ fluorophore, which can be carried either by the ligand or by the receptor. Three parameters (i) the two fluorophores have to exhibit energy compatibility; The emission spectrum of the ‘donor’ should overlap the excitation spectrum of the ‘acceptor’ and the extent of overlap directly impacts the FRET efficacy (ii) the dipole moments of the fluorophores have to be correctly oriented, the FRET being null when dipoles are perpendicular and maximum when they are parallel; and (iii) the fluorophores have to be close enough, usually less than 10 nm apart

Time Resolved FRET with Lanthanide Cryptates to Increase Signal to Noise Ratio To increase the sensitivity of FRET approaches, a new type of FRET was developed to address these issues implementing lanthanides as donor fluorophores. Whereas classical fluorophores have fluorescence lifetimes in the range of a few nanoseconds, lanthanides are characterized by much longer fluorescence lifetimes, between several hundred microseconds to a few milliseconds. This distinctiveness has led to the development of Time-Resolved FRET (TR-FRET), a technique in which the donor fluorophore is excited with a laser or a flash-lamp before measuring the emission signal

Other label free ligand assays ACOUSTIC BIOSENSOR ASSAYS Acoustic biosensors also allow the label-free detection of molecules and analysis of binding events. Based on quartz crystal resonators Linear relationship between mass adsorbed to the surface and the resonant frequency of the crystal in air or a vacuum By monitoring the change in resonant frequency and motional resistance that occurs upon adsorption of a ligand to the surface, quartz crystal resonators can be used to characterize interactions with small molecules Real time monitoring of changes in the resonant properties of the crystal allows the label-free determination of interaction affinities and kinetics (size range) Acoustic sensor response is sensitive not only to the mass of ligand bound, but also to changes in viscoelastic properties and charge of the receptor–ligand complex

Acoustic detection of visco-elastic changes The ability of quartz crystal resonator sensors (QCRS) to monitor both mass and visco-elastic changes of lipid layers and embedded receptors has allowed detailed study of the physical and chemical properties of model membrane systems and of the interactions of these systems with specific ligands Sensors can be supported with membrane layers allow the measurement of visco-elastic changes at the surface PNA T-antigen interactions: galactosyl (-1,3) N-acetylgalactosamine T antigen, viscoeleastic changes, bioadhesives Annexin-membrane interactions: -ve charge lipids, PS, Ca dependent

Quantitation of drug partitioning in model membranes The extent of passive and active adsorption of a drug to membrane interfaces in the gastrointestinal tract and blood–brain barrier is important in drug discovery Different in vitro techniques are available that measure the partitioning in membranes Immobilized lipid vesicles or cell membranes can be combined with QCRS and specific coupler detection can be used to determine the affinity of drugs for these membranes. Gives an indication of the fraction of drug passively adsorbed, and correlates well with data obtained by more traditional methods

Sedimentation assays Sedimentation and analytical ultracentrifugation are versatile tools for the study of proteins in general More quantitative data can be obtained Using lipid vesicles with defined composition and size. Multilamellar vs unilamellar vesicles: pelleting efficiency and homogeniety High density vesicles with sucrose or dextran Preparation of vesicles, incubation with protein, quantitiation of bound protein after separation Proteins with different tertiary and quaternary structures in vesicles than in lipid bilayers

Limitations: pelleting after binding, fixed concentration of lipid vesicles in the micromolar range be employed in the binding assay, which restricts its use for sub-nanomolar affinities Lengthy centrifugation can perturb the equilibrium between bound and free proteins, especially if the dissociation rates are fast, !KD values determined may not represent true equilibrium constants Peripheral proteins can be non-specifically adsorbed on the test tube walls during high-speed centrifugation.

Membrane immobilization on Analytical surfaces Supported lipid monolayers The simplest method for the immobilization of membranes on a sensor surface is to adsorb the lipid onto a hydrophobic surface This results in the formation of a supported lipid monolayer in which the hydrophobic acyl chains of the lipids contact the hydrophobic surface and polar heads face the solution limited to receptors that are anchored only in the outer leaflet of a native membrane, or to cases in which the analyte binds to the lipid itself formed from small unilamellar vesicles that spontaneously adhere to a hydrophobic self-assembled monolayer (SAM), with release of their strain energy. More hydrophilic surfaces promote the formation of lipid bilayers Lipid monolayers are highly homogeneous, possess very few surface defects and resist non-specific binding of proteins such as BSA Allows lateral diffusion of proteins, resistant to washing buffers

TETHERED BILAYER MEMBRANES A lipid layer directly adsorbed onto the surface has the significant drawback that it cannot accommodate transmembrane proteins with sizeable cytosolic or extracellular domains. To overcome this limitation several methodologies have been developed to space a lipid bilayer at some distance away from the surface They are readily formed by self assembly, are very stable Thiophospholipid possessing a triethyleneglycol spacer unit to capture membranes and membrane proteins ,membrane-spanning thiophospholipid to improve the stability of the tBLM

POLYMER SUPPORTED LIPID LAYERS These are used if surfaces are rough to accommodate the lipid monolayers the formation of lipid bilayers bound to, but structurally decoupled from the solid support by a flexible polymer. These soft polymer cushions provide a lubricating layer between the surface and the membrane, and enable the ‘self-healing’ of surface defects that increase the degree of non-specific binding to the surface dextran or hyaluronic acid, or molecules with alkyl side chains which insert into and anchor membranes Affinity captured liposomes: An alternative method to overcome problems associated with surface roughness is to derivatize a hydrogel polymer layer with a receptor that can bind to, and hence capture membranes or vesicles that possess a receptor specific ligand.