Fluorescence and FRET Concepts

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Questions and Answers

What characteristic of fluorescent molecules generally contributes to their ability to emit light?

Extensive aromatic rings (correct)

Ridge structural integrity

Low rotational state (correct)

High internal flexibility

What is the relationship between the absorption and emission spectra of a fluorescent molecule?

Emission spectra show completely different peaks from absorption spectra

Emission spectra are identical to absorption spectra

Emission spectra only occur at higher energy levels than absorption spectra

Emission spectra tend to mirror the peaks of absorption spectra (correct)

Which of the following factors affects fluorescence intensity?

Concentration of the sample

Molecular weight of the fluorophore

Type of solvent used

Excitation and emission wavelengths (correct)

What happens when a molecule with considerable internal flexibility absorbs energy?

It tends to reach the ground state without emitting light Signup and view all the answers

In a fluorescence spectrometer, what is the primary role of the emission monochromator?

To separate emitted light into its component wavelengths Signup and view all the answers

How does the sensitivity of fluorescence measurement compare to absorbance measurement?

Fluorescence is fundamentally more sensitive than absorbance Signup and view all the answers

Which of the following fluorophores is commonly used in biochemistry?

Ethidium Signup and view all the answers

What does the peak observed in a fluorescence spectrum indicate?

Particular excitation and emission wavelengths Signup and view all the answers

What is the primary mechanism by which Forster resonance energy transfer (FRET) occurs?

Direct energy transfer without photon emission Signup and view all the answers

What distance range is necessary for FRET to effectively occur?

Less than 10 nm Signup and view all the answers

Which of the following applications is NOT supported by FRET?

Tracking the pumping of ions across membranes Signup and view all the answers

In the FRET example involving SARS-CoV 3CL protease, which fluorescence was excited?

290 nm Signup and view all the answers

Which type of fluorescent dye requires cells to be permeabilized?

Modified antibodies Signup and view all the answers

What happens to the donor fluorescent intensity during FRET?

It decreases Signup and view all the answers

What is the role of Trp in FRET?

It serves as a suitable donor for common acceptors Signup and view all the answers

What type of molecules can fluorescent dyes specifically bind to in cells?

Cellular macromolecules Signup and view all the answers

Which amino acid is primarily used as a reporter of protein conformation due to its significant fluorescence?

Tryptophan Signup and view all the answers

What is the primary reason for the blue shift of the emission maximum of the NS5B protein compared to free Tryptophan?

Shielding of Trp residues from the aqueous phase Signup and view all the answers

What effect does denaturation with 8 M urea have on Tryptophan's fluorescence emission?

It results in a red shift of λmax toward 350 nm Signup and view all the answers

What can Tryptophan fluorescence be used to monitor in proteins?

Protein unfolding Signup and view all the answers

At what excitation wavelength is the LDH thermal denaturation profile monitored?

295 nm Signup and view all the answers

What characteristic of the Tryptophan emission spectrum is influenced by its environment?

Both emission intensity and wavelength of emission maximum Signup and view all the answers

What happens to the total fluorescence intensity of Tryptophan during protein denaturation?

It significantly decreases Signup and view all the answers

The emission maximum of Tryptophan in its native state is typically around which wavelength?

350 nm Signup and view all the answers

What is the absorbance maximum of DAPI?

358 nm Signup and view all the answers

What types of structures does DAPI stain in cells?

All DNA, particularly the nuclei Signup and view all the answers

What effect does phalloidin have on actin filaments?

It inhibits actin de-polymerization. Signup and view all the answers

What is the emission peak wavelength of GFP?

509 nm Signup and view all the answers

What is the main function of the cyclic peptide phalloidin?

To prevent the de-polymerization of actin filaments. Signup and view all the answers

Which creature produces green fluorescent protein (GFP)?

Pacific jellyfish Signup and view all the answers

What is the consequence of DAPI binding to DNA?

It increases the fluorescence of DNA. Signup and view all the answers

What distinguishes synthetic analogs of phalloidin from natural phalloidin?

They can be pre-labelled with various fluorophores. Signup and view all the answers

What factor is NOT directly mentioned as affecting fluorescent intensity?

Temperature of the environment Signup and view all the answers

How can extrinsic fluorophores bind to biomolecules?

Covalently and non-covalently to various biomolecules Signup and view all the answers

What is the nature of GFP in terms of fluorescence?

GFP is naturally fluorescent with a blue excitation peak. Signup and view all the answers

Which fluorophore is specifically mentioned for peptidase assays?

7-amino-4-methylcoumarin (AMC) Signup and view all the answers

What is suggested to result in higher fluorescence intensity?

Higher quantum yield Signup and view all the answers

Which structure of GFP is comprised of a beta-barrel?

An 11 stranded beta-barrel with an additional strand. Signup and view all the answers

What is the significance of Ser65 in the GFP fluorophore?

It is involved in the cyclization of the fluorophore. Signup and view all the answers

Which of the following biomolecules is known for its intrinsic fluorescence?

Tryptophan Signup and view all the answers

What is NOT a method of detecting biomolecules using fluorescence spectroscopy?

Measuring absorbance Signup and view all the answers

Which of the following statements is true regarding the expression of GFP?

GFP can be expressed recombinantly in most organisms. Signup and view all the answers

In the given analyses, what role do ‘dye-deoxy’ terminators serve?

They provide fluorescent signals for DNA sequencing. Signup and view all the answers

What role do the three residues (Ser65, Tyr66, Gly67) play in GFP?

They react to form the fluorophore. Signup and view all the answers

Which step is NOT involved in the formation of the GFP fluorophore?

Dimerization. Signup and view all the answers

What property of ClpP is associated with higher fluorescence intensity?

Higher quantum yield Signup and view all the answers

Which of the following describes the quantum yield of GFP?

It has an excellent quantum yield. Signup and view all the answers

Which statement accurately describes the behavior of intrinsic fluorescence?

It is specific to certain amino acids like tryptophan. Signup and view all the answers

In peptidase assays using AMC, what indicates the activity of ClpP?

Increased fluorescence intensity Signup and view all the answers

Which of the following properties makes GFP particularly useful in molecular biology?

Its capacity to fluoresce without the need for additional dyes. Signup and view all the answers

Study Notes

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

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Description

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.