messengers and energy sources for biochemical process. Many
nucleotide-binding proteins serve essential functional roles including translation, cellular import and export, cell signaling, biosynthesis and catabolism, cell motility, to name only a few. While there
are enzymes that bind all nucleotides present in the cell, the majority of enzymes bind and utilize either adenosine triphosphate (ATP)
or guanosine triphosphate (GTP).
Techniques to measure the interaction between a protein and
its ligands are becoming ever more important. Here we provide
examples to measure the kinetic parameters for enzymes and
nucleotides; these techniques are highly versatile and can also be
adapted for use with ligands that are not nucleotides. Besides their
critical role in drug development, these techniques are also being
used, for example, by the biotechnology industry as they continue
to develop new protein-based products for various applications [2–
4]. Several experimental approaches exist that can be used to determine the affinities, including isothermal calorimetry (ITC), nuclear
magnetic resonance (NMR), surface plasmon resonance (SPR), and
radiography-based techniques. Historically, fluorescence-based
techniques, including the methods described here, have been
used widely due to their great versatility with respect to the samples,
sensitivity, and data obtainable. Determining which technique
should be used depends on the system being examined and should
be well thought out before proceeding.
Here we describe two examples for measuring the dissociation
constant (K d ) for a protein and a ligand via fluorescence-based
techniques, both using the intrinsic fluorescent properties of the
protein and/or a fluorescently labeled ligand or protein. Primarily
this chapter will focus on assays that use two fluorophores for
Fo ¨rster/fluorescence resonance energy transfer (FRET) but can
be adapted for single fluorophore studies as well. FRET is the
nonradiative transfer of energy between an excited donor fluorophore and acceptor fluorophore in a proximity-dependent manner.
As such, FRET is a distance-dependent measurement between the
donor and acceptor fluorophores that increases the signal-to-noise
ratio by separating the excitation and emission wavelengths. Conversely, FRET does require two fluorophores (or a fluorophore and
quenching dye) that may alter the dynamics of the biomolecule
they are attached to and/or not all biomolecules are amendable for
being labeled with a fluorophore. Natural fluorophores (such as
tryptophan and tyrosine in proteins) can be used in conjunction
with synthetic fluorophores to create a FRET pair where only the
ligand needs to be labeled. Fluorescently modified analogs of certain ligands can be purchased, or produced, such as Mant-labeled
((2
0 -(or-3
0 )-O-(N-Methylanthraniloyl) nucleotides used in this
chapter to measure FRET between intrinsic tryptophan residues
in a protein (here HflX) and the Mant-fluorophore on the nucleotide (here Mant-GDPNP) for K d measurements [5, 6].
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Harland E. Brandon and Hans-Joachim Wieden
nucleotide-binding proteins serve essential functional roles including translation, cellular import and export, cell signaling, biosynthesis and catabolism, cell motility, to name only a few. While there
are enzymes that bind all nucleotides present in the cell, the majority of enzymes bind and utilize either adenosine triphosphate (ATP)
or guanosine triphosphate (GTP).
Techniques to measure the interaction between a protein and
its ligands are becoming ever more important. Here we provide
examples to measure the kinetic parameters for enzymes and
nucleotides; these techniques are highly versatile and can also be
adapted for use with ligands that are not nucleotides. Besides their
critical role in drug development, these techniques are also being
used, for example, by the biotechnology industry as they continue
to develop new protein-based products for various applications [2–
4]. Several experimental approaches exist that can be used to determine the affinities, including isothermal calorimetry (ITC), nuclear
magnetic resonance (NMR), surface plasmon resonance (SPR), and
radiography-based techniques. Historically, fluorescence-based
techniques, including the methods described here, have been
used widely due to their great versatility with respect to the samples,
sensitivity, and data obtainable. Determining which technique
should be used depends on the system being examined and should
be well thought out before proceeding.
Here we describe two examples for measuring the dissociation
constant (K d ) for a protein and a ligand via fluorescence-based
techniques, both using the intrinsic fluorescent properties of the
protein and/or a fluorescently labeled ligand or protein. Primarily
this chapter will focus on assays that use two fluorophores for
Fo ¨rster/fluorescence resonance energy transfer (FRET) but can
be adapted for single fluorophore studies as well. FRET is the
nonradiative transfer of energy between an excited donor fluorophore and acceptor fluorophore in a proximity-dependent manner.
As such, FRET is a distance-dependent measurement between the
donor and acceptor fluorophores that increases the signal-to-noise
ratio by separating the excitation and emission wavelengths. Conversely, FRET does require two fluorophores (or a fluorophore and
quenching dye) that may alter the dynamics of the biomolecule
they are attached to and/or not all biomolecules are amendable for
being labeled with a fluorophore. Natural fluorophores (such as
tryptophan and tyrosine in proteins) can be used in conjunction
with synthetic fluorophores to create a FRET pair where only the
ligand needs to be labeled. Fluorescently modified analogs of certain ligands can be purchased, or produced, such as Mant-labeled
((2
0 -(or-3
0 )-O-(N-Methylanthraniloyl) nucleotides used in this
chapter to measure FRET between intrinsic tryptophan residues
in a protein (here HflX) and the Mant-fluorophore on the nucleotide (here Mant-GDPNP) for K d measurements [5, 6].
274
Harland E. Brandon and Hans-Joachim Wieden