30
of FREX and other two-component designs is that the ratiometric
FRET change observed upon binding tends to be greater than that
of single-component sensors, because FRET efficiency is typically
reduced to near-zero values in the unbound state of the twocomponent sensors. The main limitation of FREX is that the POI
and fragment should be present at close to equimolar concentrations to achieve maximum FRET response. The protocols for creating POI-AFF and POI-FREX sensors are very similar. We outline
the protocol for AFF below, and enumerate the modifications for
FREX after each step.
2 Gathering Ingredients: The POI
1. An available X-ray or other high-resolution structures of the
POI or homolog thereof greatly facilitate the design process.
2. For AFF, the POI should ideally not contain any reduced Cys
residues, as fluorophores are typically introduced by thiolreactive chemistry. Cys in the POI may be tolerated vis-à-vis
fluorescence labeling if they are buried and inaccessible to solvent. FREX can make use of fluorescent proteins for detection
so reduced Cys residues are not inherently problematic. For
both AFF and FREX, if oxidized Cys are present, the duplicate
segment must be chosen (see Subheading 3) such that it is not
crosslinked to the nonduplicated region of the POI by a disulfide bond.
3. Consider using the most stable variant of the POI available,
e.g., one derived from a thermophilic organism. An axiom of
protein folding is that it is far easier to destabilize a protein
than to stabilize it. Accordingly, most of the modifications and
tuning mutations employed herein either intentionally or
unintentionally destabilize the POI. Starting with a stable template allows for greater design freedom.
3 Step 1 of AFF Protocol: Choosing the Segment of the POI to Duplicate
The only absolute requirement for the duplicate segment is that it
contain at least one residue that, when mutated, greatly reduces
affinity of the POI for its target ligand. Binding knockout mutations are often known from prior functional or genetic studies, and
they may also be deduced from an existing crystal structure of the
POI or homolog thereof. We have found that choosing a binding
mutation close to the beginning or end of the amino acid sequence
is advantageous, because this allows the duplicate segment to be
short in length. Since the duplicated amino acids extend from
POI-AFF as N- or C-terminal tails (Fig. 1a), shorter segments may
3.1 Identify
a Binding Mutation
Jeung-Hoi Ha and Stewart N. Loh
of FREX and other two-component designs is that the ratiometric
FRET change observed upon binding tends to be greater than that
of single-component sensors, because FRET efficiency is typically
reduced to near-zero values in the unbound state of the twocomponent sensors. The main limitation of FREX is that the POI
and fragment should be present at close to equimolar concentrations to achieve maximum FRET response. The protocols for creating POI-AFF and POI-FREX sensors are very similar. We outline
the protocol for AFF below, and enumerate the modifications for
FREX after each step.
2 Gathering Ingredients: The POI
1. An available X-ray or other high-resolution structures of the
POI or homolog thereof greatly facilitate the design process.
2. For AFF, the POI should ideally not contain any reduced Cys
residues, as fluorophores are typically introduced by thiolreactive chemistry. Cys in the POI may be tolerated vis-à-vis
fluorescence labeling if they are buried and inaccessible to solvent. FREX can make use of fluorescent proteins for detection
so reduced Cys residues are not inherently problematic. For
both AFF and FREX, if oxidized Cys are present, the duplicate
segment must be chosen (see Subheading 3) such that it is not
crosslinked to the nonduplicated region of the POI by a disulfide bond.
3. Consider using the most stable variant of the POI available,
e.g., one derived from a thermophilic organism. An axiom of
protein folding is that it is far easier to destabilize a protein
than to stabilize it. Accordingly, most of the modifications and
tuning mutations employed herein either intentionally or
unintentionally destabilize the POI. Starting with a stable template allows for greater design freedom.
3 Step 1 of AFF Protocol: Choosing the Segment of the POI to Duplicate
The only absolute requirement for the duplicate segment is that it
contain at least one residue that, when mutated, greatly reduces
affinity of the POI for its target ligand. Binding knockout mutations are often known from prior functional or genetic studies, and
they may also be deduced from an existing crystal structure of the
POI or homolog thereof. We have found that choosing a binding
mutation close to the beginning or end of the amino acid sequence
is advantageous, because this allows the duplicate segment to be
short in length. Since the duplicated amino acids extend from
POI-AFF as N- or C-terminal tails (Fig. 1a), shorter segments may
3.1 Identify
a Binding Mutation
Jeung-Hoi Ha and Stewart N. Loh