37
3 and primer 6 to the POI gene. Mix the N-frame template, N′-frame
template, primer 1, and primer 3, and generate the full- length AFF
gene using the overlap extension PCR method (Fig. 4c) [18].
Primer 1 and primer 3 will each anneal to a second, undesirable site
within the N′-frame and N-frame template, respectively, and this
results in shorter PCR products. Purify the longest PCR product
by agarose gel and subclone into the expression vector.
The cloning steps for creating FREX constructs are simpler, since
the duplicated DNA sequences are never physically joined and no
linker is present. At this stage, we find it useful to fuse the gene
of a donor and acceptor FP to the either end of the POI and fragment genes. In addition to providing a direct binding assay for
sensor tuning (Subheading 5), an FP serves as a carrier protein to
help the fragment express well, resist degradation, and stay soluble in cells.
5 Step 3 of AFF Protocol: Optimization
The objective of this step is to tune the thermodynamics of the
sensor so that it is mainly in the N-form in the absence of ligand,
and switches to the N′-form upon ligand binding. For the sake of
discussion, we assume the most common outcome of Subheading
3, i.e., that the CP form of the POI was found to be less stable than
the WT POI, and that the binding mutation was introduced into
the N-frame. The ideal distribution of N:N′ populations in the
absence of ligand is ~10:1. This scenario, which corresponds to the
N-fold being ~1.4 kcal/mol more stable than the N′-fold, achieves
an optimal balance of near-maximum fluorescence signal change
and minimal reduction of observed ligand-binding affinity
1
. Which
tuning mutation (if any) needs to be made to achieve this balance,
and into which frame it should be placed, can be predicted from
the results of Subheading 3.4. Introducing this mutation into
POI-AFF during Subheading 4 can save time by requiring less subsequent optimization in Subheading 5.
A common problem with POI-AFFs prior to their thermodynamic
balancing is that the N-fold is so much more stable than the N′fold that the ligand binds weakly or not at all. This snag is likely to
be encountered if the POI was found in Subheading 3.4 to be
much more stable than the CP (e.g., ΔT m > 10 °C or
ΔΔG unfold > 3 kcal/mol; see Subheadings 3.4.1 and 3.4.2). We
1 Because a portion of the binding energy is used to drive the N → N′ conformational change, the observed
K d will be greater than the intrinsic K d of the POI by a factor of (1 + K N +K N′ )/K N , where K = exp (‐ΔG unfold /
RT) for the respective N- and N′-folds [3].
4.4 Modifications
to Step 2 for FREX
5.1 Binding Positive
Control
Engineering Allosteric Protein Switches
3 and primer 6 to the POI gene. Mix the N-frame template, N′-frame
template, primer 1, and primer 3, and generate the full- length AFF
gene using the overlap extension PCR method (Fig. 4c) [18].
Primer 1 and primer 3 will each anneal to a second, undesirable site
within the N′-frame and N-frame template, respectively, and this
results in shorter PCR products. Purify the longest PCR product
by agarose gel and subclone into the expression vector.
The cloning steps for creating FREX constructs are simpler, since
the duplicated DNA sequences are never physically joined and no
linker is present. At this stage, we find it useful to fuse the gene
of a donor and acceptor FP to the either end of the POI and fragment genes. In addition to providing a direct binding assay for
sensor tuning (Subheading 5), an FP serves as a carrier protein to
help the fragment express well, resist degradation, and stay soluble in cells.
5 Step 3 of AFF Protocol: Optimization
The objective of this step is to tune the thermodynamics of the
sensor so that it is mainly in the N-form in the absence of ligand,
and switches to the N′-form upon ligand binding. For the sake of
discussion, we assume the most common outcome of Subheading
3, i.e., that the CP form of the POI was found to be less stable than
the WT POI, and that the binding mutation was introduced into
the N-frame. The ideal distribution of N:N′ populations in the
absence of ligand is ~10:1. This scenario, which corresponds to the
N-fold being ~1.4 kcal/mol more stable than the N′-fold, achieves
an optimal balance of near-maximum fluorescence signal change
and minimal reduction of observed ligand-binding affinity
1
. Which
tuning mutation (if any) needs to be made to achieve this balance,
and into which frame it should be placed, can be predicted from
the results of Subheading 3.4. Introducing this mutation into
POI-AFF during Subheading 4 can save time by requiring less subsequent optimization in Subheading 5.
A common problem with POI-AFFs prior to their thermodynamic
balancing is that the N-fold is so much more stable than the N′fold that the ligand binds weakly or not at all. This snag is likely to
be encountered if the POI was found in Subheading 3.4 to be
much more stable than the CP (e.g., ΔT m > 10 °C or
ΔΔG unfold > 3 kcal/mol; see Subheadings 3.4.1 and 3.4.2). We
1 Because a portion of the binding energy is used to drive the N → N′ conformational change, the observed
K d will be greater than the intrinsic K d of the POI by a factor of (1 + K N +K N′ )/K N , where K = exp (‐ΔG unfold /
RT) for the respective N- and N′-folds [3].
4.4 Modifications
to Step 2 for FREX
5.1 Binding Positive
Control
Engineering Allosteric Protein Switches