Protein-Ligand Binding_L3 Flashcards
principles of the flurescence method
(1) . Fluorescent molecules (fluorophores) absorb light of one colour (wavelength) and emit the energy at a different wavelength (colour)
(2) . Fluorophores act as visual probes. They may be intrinsic (e.g. Trp and amino acid containing the aromatic ring structure such as the phenylalanine), can be covalently attached(use some chemical handles) to ligands or remain in solution.
(3) . Fluorescence is sensitive to environmental changes (e.g. from hydrophilic to hydrophobic) – much more so than absorption:
1. Fluorescence increases in hydrophobic, ‘rigid’ environments
2. Collisions of a fluorophore with solvent molecules leads to decay of excited states without light emission – quenching, e.g. ethidium bromide is quenched in aqueous solvent – but not when bound to DNA
How are the flurescence and energy level involved in fluorescence method?
(1) absoption: the ground state transition to the excited state with vibrational levels
(2) radicational decay to the lowest vibrational level in the excited state
(3) transition to the grounf state with the fluorescence emission and some energy is lost in the heat form: the emitted light is of lower energy than the absorbed light, and therefore lower wavelength
What is fluorescein and what characteristics does the fluorephore have?
(1) Absorption max at 494 nm. Emission max at 523 nm
(2) fluorescein is a fluorephore
(3) the molecular structure contains vast number of ring structure and pi electrons
(4) the fluorescence is emitted in all direactions
What is fluorescence resonance energy transfer(FRET) and what theories it is based on?
(1) Interaction between the excited states of two fluorescent molecules. Used to detect proximity between two fluorophore-labeled biomolecules.
(2) Excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon: the spectrum of the donar molecule overlaps partially with the spectrum of the acceptor molecule
(3) Donor and acceptor molecules must be close together (10 to 60 Å) - so method can report on binding, no direct physical contact is required.
What real data will be resulted from the FRET experiement?
If the donar molecule is CFP which can be excited at 340 nm, the fluorescence it yielded emits at 475-500 nm, which in turn excites the acceptor protein YFP to fluoresce at 525-550nm
donar accepted-> donar emitted->acceptor accepted wavelength same as the donar emitted wavelength if the two proteins are in interaction or in close proximity->acceptor emits
- CFP = cyan fluorescent protein
- YFP = yellow fluorescent protein
the fluorimeter setup?
The light source emits directly through the monochromator to the cuvette containing the sample which is placed in the vertical line as the light source and the monochromator. Another monochromator is set at 90 degrees from the incident beam to ensure that all the measured beams are orginated from the sample, at the back of the monochromator is the detector for measuring the wavelength of the emitted light.
Monochromators allow the excitation light wavelength to be selected and the collect the light emitted from the sample.
The equation for measuring the fluorescent intensity?
Y=(F-F0)/(Fmax-F0)
Y: fractional saturation
F: the intensity measured from the sample
F0: the intensity when no ligand is bound
Fmax: the maximum intensity when the proteins are fully saturated
In this experiment, the proteins are fluorescently labelled but the ligands are left unlabelled, the saturation equation can therefore be applied with the independent variables being replaced with the [P]:
Y=[P]/([P]+Kd) where the Kd is constant.
What is differential scanning fluorimetry(DSF) and what theories it is based on?
(1) Methodologically simple technique – qPCR/rtPCR machine
(2) Relies on changes in thermal stability of a protein upon ligand binding, the fluorescence is measured as the experiment progresses.
(3) Useful to test binding of peptides and nucleic acids, sugars(for some reasons, the technique works extremely accurate for the nucleic acids)
(4) Useful to optimise solution conditions of a protein under study – buffer, ionic strength, cofactors, etc.
(5) Ideal for screening of libraries of small MW ligands
– widely used in FBDD(fragment-based drug discovery)
What does the DSF fluorescence graph inform us of?
(1) Tm: melting temperature is the most critical parameter in the experiment, measuring the protein thermal stability
(2) the curve starts with relatively flat line representing the minimal interaction of the soluble dye to the protein in the native state.
(3) The line goes up to the climate where the protein-dye interaction is the greatest since the hydrophobic patches are exposed to the dye binding.
(4) The furrther increase in temperaturer does not strengthen the interaction any further due to the reason that the protein aggregation is more favourable with the denaturation progressing.
(5) DSF compares the wild-type(WT) proteins with the mutants as well as the WT protein-ligand complex with the mutant-ligand complex.
pros and cons of DSF
(1) pros
1. Inexpensive and quick: 96 experirments can be conducted in couple of hours
2. High throughput
3. Easy to visualise the result
4. Identification of weak interactions
5. semi-quantitative
6. Can compare different proteins/mutants easily
7. Widely applicable – does not rely on intrinsic fluorescence of a protein: the due can be fused onto the protein of interest
(2) cons:
1. False negatives can be observed (FBDD) – use secondary techniques
2. Small or elongated proteins with reduced hydrophobic core
3. Fluorescent ligands can affect the fluorescent intensity
What are the priciples of microscale thermophoresis?
(1) Principle: thermophoresis is the directed movement of molecules in a temperature gradient
- An increase in temperature at centre of IR irradiation (the detected area), will rapidly drive away fluorescent molecules, e.g. small ligands (yellow) – large fluorescence change(ligand moves rapidly).
- At different points of the titration, increasing concentrations of unlabeled binding partner (e.g. large protein, red), will slow down migration of bound fluorescent ligand – smaller fluorescence change(ligands bound to larger molecule/protein migrates in slower rate).
(2) IR(infrared) laser radiation is focused on specific point at sample in a capillary tube
(3) Fluorescent signal change is generated from a volume of ~2nL that is heated 1-6 ⁰C
(4) Experiments involve 16 samples with serial dilution of unlabelled binding partner (i.e. with the largest MW)
(5) Determination of KD in ~30min(suitable systems)
(6) Any change of the hydration shell of biomolecules due to changes in their primary, secondary, tertiary and/or quaternary structure affects the thermophoretic
movement and is used to determine binding affinities
with high accuracy and sensitivity.
What is the experimental setup of the microscale thermophoresis?
(1) infrared (IR) light reflected from the hot mirror and is focused by the objective lens to hit on the capillary containing the sample
(2) fluorescent light at 90 angle incidents to the sample tray
(3) the reflected flourescent light from the sample is emitted out as the fluorescent observation
(4) According to the fluorescent intensity, the plot is obtained as the change in fluorescent intensity versus the timescale over the molecular migration, the more concentrated the proteins are the smaller distance of migration, the intensity gap between the two extremes: hot and cold extremes will be reduced.
(5) By translating the intensity vs time graph into the Δintensity vs ligand concentration , the plot is more informative
pros and cons of microscale thermophoresis
pros:
1. Highly sensitive, quantitative analysis of protein interactions
2. Analysis in solution, no immobilization of ligands and target proteins
3. Low sample consumption: a series of capillary tubes with increasing ligand concentrations
4. Fluorescent labelling and intrinsic UV-fluorescence (in suitable systems)
5. Very fast determination of KD’s
cons:
1. Labelling of reagents?
2. Applicable to all systems?
3. Principle of thermophoresis not well understood in solution
→ Limited use as an ‘orthogonal’ technique?
4. Throughput for screening of ligands?
5. Upper limit of KD restricted by solubility of high MW binding partner?
pros and cons of fluorescent method
(1) Sensitive
(2) Can be quantitative
(3) Equilibrium measurements (in vitro)
(4) Use to examine protein-protein interactions in living cells
(5) May require special reagents
(6) GFP-fusion proteins
(7) Chemical attachment of fluorophores to your protein
