Transcript labelled ligand assays_1 and 2

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) Nacetylgalactosamine 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 ,membranespanning 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 ‘selfhealing’ of surface defects that increase the degree of nonspecific 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.