Protein-Protein Interaction Analysis Using Fluorescence Spectroscopy: Practical Examples
Masoud Shaygan1, Hossein Ahmadzadeh1*
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
*To whom correspondence should be addressed: h.ahmadzadeh@um.ac.ir
Abstract:
This chapter provides an in-depth exploration of the application of fluorescence spectroscopy in
studying protein-protein interactions. Fluorescence spectroscopy offers a powerful and versatile
approach to investigate the dynamic nature of protein-protein interactions, providing valuable insights
into their mechanisms, kinetics, and thermodynamics. This chapter presents fundamental principles,
experimental techniques, and practical examples of fluorescence spectroscopy methods employed in
protein-protein interaction studies. Additionally, it highlights the importance of careful experimental
design, data analysis, and interpretation of results.
1. Introduction
Protein-protein interactions (PPIs) are physical contacts of high specificity established between two
or more protein molecules as a result of biochemical events steered by interactions that include
electrostatic forces, hydrogen bonding and the hydrophobic effect. PPIs are essential for many
biological processes and molecular machines that are built from numerous protein components
organized by their PPIs. However, PPIs are also challenging to study and verify, as they can be
affected by false positives, false negatives, and technical artifacts. Therefore, researchers should
carefully choose the most suitable PPI method for their research question, optimize the experimental
conditions, validate the interactions by independent methods, and compare the results with existing
databases and literature.
Fluorescence spectroscopy is one of the most widely used and powerful techniques to analyze PPIs,
as it can provide information about the structure, dynamics, and function of interacting proteins.
Fluorescence spectroscopy is based on the principle that fluorescent molecules can absorb and emit
light of specific wavelengths, and that their fluorescence properties can change upon interaction with
other molecules. By using fluorescent probes that are attached to the proteins of interest, researchers
can monitor the changes in fluorescence intensity, lifetime, spectrum, polarization, or energy transfer
that reflect the occurrence and characteristics of PPIs.
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This chapter aims to provide a comprehensive overview of the application of fluorescence
spectroscopy in studying PPIs. It covers the basic concepts and principles of fluorescence
spectroscopy, the experimental techniques and methods that are commonly used for PPI analysis, and
the practical examples and case studies that illustrate the advantages and limitations of fluorescence
spectroscopy in PPI research. The chapter also emphasizes the importance of careful experimental
design, data analysis, and interpretation of results, as well as the integration of fluorescence
spectroscopy with other complementary techniques.
2. Principles of Fluorescence Spectroscopy
2.1 Fluorescence Phenomenon and Basic Concepts
Fluorescence is the phenomenon of light emission by a molecule that has previously absorbed light
of a higher energy. When a molecule absorbs a photon of light, it undergoes a transition from the
ground state to an excited state. The excited state is usually unstable and short-lived, and the molecule
can return to the ground state by various pathways. One of these pathways is fluorescence emission,
which involves the release of a photon of lower energy than the absorbed one. The difference in
energy between the absorbed and emitted photons is called the Stokes shift, and it is usually dissipated
as heat or vibrational energy.
The fluorescence emission of a molecule depends on several factors, such as the absorption and
emission spectra, the quantum yield, the lifetime, the quenching mechanisms, and the environmental
conditions. The absorption spectrum of a molecule is the plot of the absorption coefficient versus the
wavelength of the incident light, and it shows the range of wavelengths that can excite the molecule.
The emission spectrum of a molecule is the plot of the fluorescence intensity versus the wavelength
of the emitted light, and it shows the range of wavelengths that the molecule can emit. The quantum
yield of a molecule is the ratio of the number of photons emitted to the number of photons absorbed,
and it indicates the efficiency of the fluorescence process. The lifetime of a molecule is the average
time that the molecule spends in the excited state before returning to the ground state, and it reflects
the rate of the fluorescence decay. The quenching mechanisms of a molecule are the processes that
reduce the fluorescence intensity or lifetime by transferring the excess energy to other molecules or
pathways, such as collisional quenching, static quenching, or non-radiative decay. The environmental
conditions of a molecule are the factors that affect the fluorescence properties by altering the
electronic structure or the conformation of the molecule, such as temperature, pH, solvent, or binding
partners.
2.2 Fluorophores and Their Properties
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Fluorophores are molecules that can fluoresce when excited by light. Fluorophores can be classified
into two types: intrinsic and extrinsic. Intrinsic fluorophores are molecules that are naturally present
in biological samples, such as amino acids, cofactors, or nucleic acids. Extrinsic fluorophores are
molecules that are artificially introduced into biological samples, such as synthetic dyes, proteins, or
nanoparticles. Fluorophores can be attached to the proteins of interest by various methods, such as
covalent bonding, non-covalent binding, genetic fusion, or physical incorporation.
Fluorophores have different properties that determine their suitability and performance for
fluorescence spectroscopy applications. Some of the important properties are:
- **Spectral characteristics**: These include the absorption and emission spectra, the Stokes shift,
and the spectral overlap. The spectral characteristics of a fluorophore determine the wavelength of
the excitation and emission light, the amount of energy loss, and the degree of interference with other
fluorophores or background signals.
- **Quantum yield**: This is the ratio of the number of photons emitted to the number of photons
absorbed, and it indicates the efficiency of the fluorescence process. The quantum yield of a
fluorophore depends on the quenching mechanisms and the environmental conditions, and it affects
the brightness and sensitivity of the fluorescence signal.
- **Lifetime**: This is the average time that the fluorophore spends in the excited state before
returning to the ground state, and it reflects the rate of the fluorescence decay. The lifetime of a
fluorophore depends on the quenching mechanisms and the environmental conditions, and it affects
the temporal resolution and contrast of the fluorescence signal.
- **Photostability**: This is the ability of the fluorophore to resist photobleaching or photodamage,
which are the processes that reduce the fluorescence intensity or alter the fluorescence properties by
exposure to light. The photostability of a fluorophore depends on the chemical structure and the
environmental conditions, and it affects the durability and reliability of the fluorescence signal.
- **Biocompatibility**: This is the ability of the fluorophore to interact with biological samples
without causing adverse effects, such as toxicity, immunogenicity, or perturbation. The
biocompatibility of a fluorophore depends on the chemical structure and the attachment method, and
it affects the safety and validity of the fluorescence signal.
2.3 Fluorescence Emission Spectra and Stokes Shift
The fluorescence emission spectrum of a fluorophore is the plot of the fluorescence intensity versus
the wavelength of the emitted light, and it shows the range of wavelengths that the fluorophore can
emit. The fluorescence emission spectrum of a fluorophore is usually broader and shifted to longer
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wavelengths than the absorption spectrum, due to the Stokes shift. The Stokes shift is the difference
in energy between the absorbed and emitted photons, and it is usually dissipated as heat or vibrational
energy.
The Stokes shift of a fluorophore is an important parameter for fluorescence spectroscopy, as it
determines the wavelength of the emission light and the degree of separation from the excitation light.
A large Stokes shift is desirable for fluorescence spectroscopy, as it allows the use of a single light
source for multiple fluorophores, reduces the interference of the excitation light with the emission
light, and enhances the detection of the fluorescence signal. However, a large Stokes shift also implies
a large energy loss, which can reduce the quantum yield and the brightness of the fluorophore.
The Stokes shift of a fluorophore depends on several factors, such as the electronic structure, the
vibrational levels, the solvent effect, and the interaction with other molecules. The electronic structure
of a fluorophore determines the energy levels and the transitions of the electrons between the ground
state and the excited state. The vibrational levels of a fluorophore are the discrete energy states of the
molecular vibrations within the electronic states. The solvent effect of a fluorophore is the influence
of the surrounding medium on the polarity and the relaxation of the fluorophore. The interaction with
other molecules of a fluorophore is the effect of the binding or the collision of the fluorophore with
other molecules that can alter the fluorescence properties.
2.4 Fluorescence Lifetime and Quenching
The fluorescence lifetime of a fluorophore is the average time that the fluorophore spends in the
excited state before returning to the ground state, and it reflects the rate of the fluorescence decay.
The fluorescence lifetime of a fluorophore is usually measured by using time-resolved fluorescence
spectroscopy, which involves the excitation of the fluorophore by a short pulse of light and the
detection of the fluorescence signal as a function of time. The fluorescence lifetime of a fluorophore
can be described by an exponential decay function, such as:
$$I(t) = I_0 e^{-t/\tau}$$
where $I(t)$ is the fluorescence intensity at time $t$, $I_0$ is the initial fluorescence intensity, and
$\tau$ is the fluorescence lifetime. The fluorescence lifetime of a fluorophore can also be described
by a multi-exponential decay function, if the fluorophore has multiple decay pathways or components,
such as:
$$I(t) = \sum_{i=1}^n \
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