Protein Fluorescence Lecture Notes

Protein Fluorescence

Further Reading

• Textbooks available via the library:

  • Exploring Proteins by Price & Nairn (2009), Oxford University Press.

  • How Proteins Work by Williamson (2011), Garland Science.

  • Introduction to Protein Science by Lesk (2010, 2nd ed.), Oxford University Press.

Physical Principle (Jablonski Diagram)

• Electron Promotion: An electron is promoted to an excited state (higher energy orbital) upon the absorbance of a photon.

• Fluorescence Lifetime: Ranges from 1-10 nanoseconds (ns).

• Fluorescence Process: Occurs when the electron transitions from the lowest vibrational level of the excited state to the ground state.

• Energy Loss: Energy is lost as heat during non-radiative transitions.

• Energy Levels

  • Ground State (S0)

  • Excited State (S1)

  • The energy of fluorescence is always lower than the absorbed light, resulting in a redshift.

Fluorescence Characteristics

• Wavelength Shift: Fluorescence occurs at a longer wavelength due to energy loss as heat (relationship: $E ext{ proportional to } 1/ ext{λ}$).

• Stokes Shift: The difference between the maximum wavelength of absorbance and emission. A larger Stokes shift facilitates easier measurement of emission without interference from excitation light.

• Fluorescence Lifetime: Average duration a fluorophore remains in an excited state before emitting a photon, typically 1-10 ns for proteins.

• Orientation Dependence: Excitation is more likely when the dipole moment of the electron transition aligns with the electric vector of the exciting light.

• Fluorescence Anisotropy: Measured based on the difference in polarization between exciting (absorbed) and emitted light, which allows measuring rotational movement of the fluorophore.

Absorbance and Polarization

• Absorbance Dependency: Depends on the angle (θ) between the transition dipole moment of the electron and the polarization of applied radiation.

  • At $θ = 0^ ext{o}$: Maximum absorption.

  • At $θ = 90^ ext{o}$: Zero absorption.

• Polarization of Emitted Light: Parallel to the transition dipole moment of the electron returning to the ground state.

Fluorimeter Functionality

• Monochromators: Allow selection of single wavelengths or scanning of a wavelength range.

• Slits Control: The amount of light reaching the sample and the detector.

• Measurement Angle: Fluorescence is measured at 90 degrees to the incident beam, enhancing sensitivity compared to absorbance spectroscopy (sensitivity increase by x100 - x1000 due to dark background).

• Sample Requirements: Samples must be free of particles to avoid light scattering; high-quality reagents are necessary to prevent high-background fluorescence from impurities.

  • Components:

  • Light Source

  • Excitation Monochromator

  • Cuvette

  • Absorbance/Emission Monochromator

  • Detector

Types of Fluorescence

• Intrinsic Fluorescence:

  • Arises from naturally occurring chemical groups in proteins, including:

  • Phenylalanine (Phe)

  • Tyrosine (Tyr)

  • Tryptophan (Trp)

  • Some co-factors like NADH

• Extrinsic Fluorescence:

  • Emerges from externally added chemical groups or fusion with naturally fluorescent proteins like GFP.

Properties of Amino Acids

Absorbance and Quantum Yield Data

• Only Tyr and Trp significantly fluoresce in proteins.

• Trp fluorescence is more intense and dominates in proteins with both Tyr and Trp due to:

1. Intensity of Trp fluorescence.

2. Energy transfer from Tyr to Trp (FRET).

Emission Spectra

• Emission spectra for Phe, Tyr, and Trp show varying amino acid concentrations, measured in arbitrary units (0-1000).

Quenching Effects on Fluorescence

• Fluorescent Intensity: Dependent on the degree of quenching of the Trp sidechain.

• Quenching Agents:

  • Internal groups such as carboxylate ($COO^{-}$), histidine, aromatic sidechains, and disulfide bonds.

  • External quenchers, such as acrylamide and iodide ($I^{-}$) ions, help investigate Trp accessibility to solvents.

• Shifts in $l_{max}$:

  • Polar environment: $l_{max} = 355 ext{ nm}$

  • Hydrophobic environment: $l_{max} = 320 ext{ nm}$

  • Changes in Trp fluorescence can report on protein folding/unfolding due to sensitivity to micro-environment.

Energy Transfer between Fluorophores

• Energy transfer occurs when the emission spectrum of the donor overlaps with the absorbance spectrum of the acceptor. Conditions include:

  • Distance: 10-100 Å between fluorophores.

  • Orientation: Dipoles must be aligned (parallel).

• Transitions:

  • Coupled transitions.

  • Radiative transitions and radiationless transitions.

• Efficiency of Energy Transfer: Depends on spectral overlap.

• Fluorescence Resonance Energy Transfer (FRET):

  • Used to measure distances within or between proteins.

  • Donor must be fluorescent; acceptor can be either fluorescent or non-fluorescent.

  • Energy transfer observed through decreases in donor fluorescence and/or increases in acceptor fluorescence.

  • Energy Transfer Efficiency Formula: $E = \frac{R<em>{0}^{6}}{R</em>{0}^{6} + R^{6}}$, where $R$ is the distance between donor and acceptor, and $R_{0}$ is the Förster distance (distance at which transfer is 50% efficient).

  • The efficiency of transfer is inversely proportional to the sixth power of distance: $E \propto \frac{1}{R^{6}}$.

  • Effective measurement requires donor and acceptor to be within $0.5<em>R{0}$ and $1.5R</em>{0}$.

Examples of FRET Pairs

• Donors:

  • Fluorescein

  • IAEDANS

  • EDANS

• Acceptors:

  • Tetramethylrhodamine

  • Fluorescein

  • DABCYL

  • QSY7 and QSY9 dyes

• Tyr → Trp Energy Transfer: Enables efficient energy transfer; amino acids must be within 20 Å (Ro = 21 Å) for efficiency.

• Covalent Attachment: Donors and acceptors often bonded through covalent ties, particularly via functional groups modifying cysteine (Cys).

Advanced Topics in Fluorescence

New Fluorescent Probes

• Development of fluorescent probes that are:

  • Bright (high absorbance and quantum yields)

  • Photo-stable.

• Example: Alexa Fluor dyes from Molecular Probes.

Biological Applications

1. Ligand Binding

• Example: Peptide EGLPPPYTV binding to WW domains demonstrated.

  • Measurement changes Trp fluorescence as ligand concentration increases.

  • Dissociation Constant (Kd) calculated from binding curves, dependent on binding constant (K) and free ligand concentration: $K_{d} = \frac{1}{K}$.

  • Low amounts of protein and peptide ligand needed (μM range), without immobilization or chemical modification.

2. Protein Denaturation and Stability

• Intrinsic Fluorescence: An ideal technique to monitor protein denaturation, fast, and requires minimal protein.

• Fluorescence spectra show N-terminal domain of TIMP-2 in native (0M GdmCl) versus denaturing (6M GdmCl) conditions.

• Denaturation Curves: Generated from changes in fluorescence at single wavelengths, indicating stability of various proteins.

3. Measurement of Intra- or Intermolecular Distances

• Example of E. coli Adenylate Kinase structural flexibility illustrated by FRET under different substrate binding conditions.

4. Assay Development

• Matrix Metalloproteinase Activity Assay Example: Quenching of Mca by FRET shows fluorescence increases with distance as FRET is prevented.

5. Detection of Hydrophobic Surfaces

• Fluorescent Dyes: Used to characterize protein structure.

  • Non-fluorescent in aqueous solutions, fluoresce when bound to hydrophobic surfaces.

• Example: ANS (1-aniline-8-naphthalene sulfonate) binding illustrated:

  • Fluorescent Changes: ANS quenched in aqueous, shows 20-fold increase upon binding to hydrophobic sites (test example: porcine somatotrophin).

  • No fluorescence increase observed with bovine pancreatic ribonuclease, indicating surface hydrophobicity differences.