Fluorescence and FRET Concepts

Fluorescence Spectroscopy

• Fluorescence spectroscopy measures the intensity of light emitted by certain molecules called fluorophores
• This emitted light has a longer wavelength than the absorbed light and is dispersed in all directions
• All fluorophores absorb light, but only some emit significant fluorescence

Essence of "-escences"

• Absorbance: A photon excites an electron to a higher energy state. The energy is then transferred as heat through rotational/translational motions
• Fluorescence: A photon excites an electron and energy is emitted as a photon in a few nanoseconds
• Phosphorescence: A photon excites an electron and energy is emitted as a photon in seconds to minutes
• Chemiluminescence: A chemical reaction excites an electron and energy is emitted as a photon, for example Luminol + H2O2
• Bioluminescence: A form of chemiluminescence in a living organism

Fluorescence

• Fluorescence measurements detect the intensity of light emitted by molecules
• The emitted light wave length is longer than the absorbed light wavelength
• All fluorophores absorb light, but only some significant fluorophores emit fluorescence

The Fluorescence Process

• When a photon is absorbed, an electron is excited to a higher energy electronic level (S₂)
• Intermolecular collisions in solution rapidly cause relaxation to the lowest vibration-rotation energy state (S₁) of the excited electronic state (S₁)
• Fluorescence emission occurs as the excited molecule returns to the lower vibrational state of the ground electronic state (S₀)
• The emission spectrum is shifted to longer wavelengths than the excitation spectrum. This is called the Stokes shift

Fluorescent Molecules

• Molecules with internal flexibility can disperse energy by transferring it into rotational modes
• This results in overlap of the highest rotational modes of the ground electronic state with the lowest excited electronic state
• These types of molecules can avoid emitting a UV/visible photon
• Rigid molecules, often polycyclic aromatics, have few rotational states to disperse energy into
• Relaxation of these molecules requires emitting a high-energy photon or fluorescence

Absorption vs Emission Spectra

• Different excited states are split by the same relaxation modes
• This means the fluorescent spectrum shows a similar pattern to the absorption spectrum, but mirrored

Examples of Fluorophores in Biochemistry

• Ethidium: A molecular structure
• Fluorescein: A molecular structure
• ANS (8-anilinonaphthalene-1-sulphonic acid): A molecular structure
• Acridine orange: A molecular structure

Fluorescence Intensity

• Fluorescence intensity is dependent on both excitation and emission wavelengths

Fluorescence Spectrometer

• Fluorescence measurements are taken relative to a dark background, which makes fluorescence more sensitive than absorbance
• A fluorescence spectrometer uses a lamp, excitation monochromator, sample cell, emission monochromator, detector to measure emitted light

Light Source: Xenon Arc Lamp

• Xenon arc lamps produce high light intensity, essential for fluorescence measurements
• These lamps use a fused quartz envelope, tungsten electrodes and are filled with xenon gas at high pressure
• The housing of the lamp protects users from explosion and bright light

Fluorescence Spectroscopy: Quantitation

• Fluorescence intensity depends directly on lamp intensity
• Fluorescence is measured by arbitrary units (a.u) in contrast to absorbance measurements
• Quantification of concentrations is possible via standard curves

Quantum Yield and Quenching

• Quantum yield (Q) is the number of emitted fluorescence photons per absorption event of light
• A molecule's environment impacts its quantum yield; interactions with solvents can quench them
• Q can be used to study the local environment of a fluorescent species

Fluorescence Spectroscopy: Further Quantitation

• Fluorescence intensity is proportional to incident light intensity, path length, concentration, quantum yield, and the molar extinction coefficient of the fluorophore

Practice Questions - Fluorescence

• The intensity of observed fluorescence is affected by the wavelength of incident light, molar extinction coefficient, incident light intensity, and concentration of the fluorophore

Applications of Fluorescence Spectroscopy in Biochemical Analysis

• Enzyme Assays: Used in peptidase/protease degradation assays.
• Biomolecule Detection: extrinsic fluorophores are covalently bound or non-covalently bound to biomolecules.
• Intrinsic Fluorescence: Detection of intrinsic fluorescence by biomolecules like tryptophan.
• Characterizing Rapid Mixing Devices: This involves submillisecond protein folding by monitoring via rapid mixing and mass spectrometry
• Monitoring Protein Transitions: This can utilize CD or Circular Dichroism spectroscopy and pulsed oxidative methods

Intrinsic Fluorescence of Polypeptides

• Tryptophan is the only amino acid with significant fluorescence
• Trp used as a reporter of protein conformation due to the sensitivity of its fluorescence to the local environment

Tryptophan Emission Spectrum

• Fluorescence intensity of Trp is highly influenced by its environment
• A shift in emission maximum will correlate with a change in the protein conformation and/or structure, which in turn allows monitoring of protein unfolding or refolding.

Protein Denaturation

• Trp fluorescence is useful for monitoring protein denaturation by measuring the changes in emission intensity or emission maximum as the denaturation process takes place during changes in temperature.

Trp Fluorescence: Structural Changes

• Carbonic anhydrase can only be active when there is a disulfide bond and it is in a reduced environment
• Trp fluorescence can monitor local structural changes in the protein.

CD Monitors Protein Structural Transitions

• CD spectroscopy is used to map pH-induced structural changes in proteins, revealing equilibrium conditions by pulsed oxidative labeling and mass spectrometry

Fluorescence and Ligand Binding

• Surface-exposed tryptophan residues are prone to quenching by the solvent
• If a Trp residue is in a binding site, ligand binding can cause increased Trp fluorescence by protection of the residue and can be used to calculate protein binding constants
• This increase in fluorescence can be used to measure the affinity of lactose to Galectin-3

Labeling Proteins with Extrinsic Fluorophores

• Maleimide groups efficiently react with thiols, forming covalent bonds.
• Maleimide groups can selectively label cysteines, and lysines by using isothiocyanate linked fluorophores.
• This can label proteins in a way that can be detected by fluorescent methods or spectrometry.

Forster Resonance Energy Transfer (FRET)

• FRET occurs when the emission spectrum of one fluorophore (donor) overlaps with the absorption spectrum of another (acceptor)
• Energy is transferred from donor to acceptor without emitting a photon, reducing donor fluorescence intensity
• FRET occurs over short distances (< 10 nm) and falls off rapidly as distance increases.

###FRET Reports on Closeness of Donor and Acceptor

• FRET is used to measure distances on a molecular scale, monitor protein complex formation, bindings, and monitor motions.

FRET Example

• FRET can be used to monitor the binding of flavonoid compounds to the SARS-CoV 3CLpro protease

Fluorescent Microscopy

• Some molecules specifically bind to cellular macromolecules
• Synthesized molecules can attach fluorescent labels
• These stains can be used to label proteins and cellular molecules, however, cells need to be permeabilized

Stain Examples

• DAPI: Binds specifically to dsDNA, stains DNA blue, useful in cell biology.
• Phalloidin: Binds tightly to actin filaments. This prevents actin depolymerization, making a wide array of useful staining tools.

Fluorescent Microscopy: Green Fluorescent Protein (GFP)

• Pacific jellyfish and similar organisms produce proteins that are bioluminescent.
• The light from these organisms can be converted to green by the protein aqeouorin
• GFP is useful because it is naturally fluorescent
• GFP has a small size that readily expresses in almost any organism
• GFP structure is a 11 stranded beta barrel and there are 3 amino acid residues in the middle that create the fluorophore.
• Engineered GFP variants allow labelling across the spectrum of light

GFP Labelling Proteins

• A strategy used is to fuse a protein of interest to a GFP variant which is useful in cell biology to monitor real time protein localization in living cells.

Practice Questions - Additional

• A benefit from using GFP to study cells instead of antibodies is that the cells can remain alive as this method does not require permeabilization.

Fluorescence Spectroscopy Summary

• Molecules absorb photons and excite electrons in molecular orbitals, which then decay.
• Fluorescence spectroscopy is used for quantitative analysis of molecules that contain fluorophores.
• Fluorescent molecules are typically structurally rigid aromatic molecules.
• Strengths: high sensitivity, high-throughput method, direct quantification
• Weaknesses: limited to molecules with good fluorophores, sensitive to quenching

Test your understanding of fluorescence principles and Forster resonance energy transfer (FRET) with this quiz. Explore topics including fluorescence intensity factors, the relationship between absorption and emission spectra, and applications of fluorophores. Perfect for students studying biochemistry or spectroscopy.