39
necessary and the sensor is ready for use. Result (ii) suggests that the
N-fold is too stable and that a moderately destabilizing mutation
should be introduced into the N-frame. If Δθ is close to zero
(outcome (iii)) then one can conclude that POI-AFF either never
switched conformation (i.e., it was in the N′-fold even in the
absence of ligand), or that it changed conformation and the fluorophores did not report on the change. In the former scenario the
solution is to introduce tuning mutations into the N′-frame
instead of the N-frame. The expectation is that Δθ will initially
increase and then approach a maximum value as the N′-fold is
progressively destabilized by packing mutations of increasing
severity [3]. Any one of these variants can be used for sensing,
although for the typical case in which a balance between high Δθ
and low K d is desired, the mutant that yields outcome (i) is selected
as the final optimized product.
The AFF sensing mechanism is reversible and the speed at which
the forward and reverse conformational changes occur determines
how rapidly the sensor can detect fluctuations in analyte concentration. It may be desirable in some applications to increase this
response rate. For calbindin D 9k and RBP, the rate limiting step of
the N→N′ and N′→N reactions appears to involve a local but not
global unfolding event, most likely unfolding/dissociation of the
copy of the duplicate segment that is docked to the shared segment
of the protein. If this result is general, any mutation that destabilizes the interaction between the shared and duplicate regions has
the potential to accelerate the conformational switch. If one wishes
to attempt to engineer a faster AFF sensor, we recommend first
following all of the thermodynamic balancing procedures described
above, then introducing kinetic tuning mutation(s) in a manner
that does not perturb the existing N/N′ balance or ligand-binding
affinity. For example, consider a hypothetical 200-amino acid POI
from which residues 150–200 were duplicated to generate POI- AFF.
Further suppose that residues Phe130 and Leu170 pack tightly
against each other. Kinetic tuning could consist of a single
Phe130→Ala substitution in the shared region, or Leu170→Ala
+ Leu170′→Ala mutations in the respective duplicate segments.
In either case the N-fold and N′-fold are in principle destabilized
equally but the kinetic barrier to their interconversion may be
lowered, depending on the extent to which the Phe130-Leu170/
Leu170′ interaction is formed in the transition state ensemble.
As noted, it is simplest to fuse donor and acceptor FPs to the fulllength POI and duplicate fragment at the cloning stage; however, if
chemical fluorophores are desired then the labeling procedure is
identical to that of AFF. One difference of FREX is that the two components of the sensor are on average very far apart in the absence of
ligand (at reasonable protein concentrations), so donor/acceptor
pairs with a wide range of Förster distances can be considered.
5.3 Optional:
Kinetic Tuning
5.4 Modifications
to Step 3 for FREX
Engineering Allosteric Protein Switches
necessary and the sensor is ready for use. Result (ii) suggests that the
N-fold is too stable and that a moderately destabilizing mutation
should be introduced into the N-frame. If Δθ is close to zero
(outcome (iii)) then one can conclude that POI-AFF either never
switched conformation (i.e., it was in the N′-fold even in the
absence of ligand), or that it changed conformation and the fluorophores did not report on the change. In the former scenario the
solution is to introduce tuning mutations into the N′-frame
instead of the N-frame. The expectation is that Δθ will initially
increase and then approach a maximum value as the N′-fold is
progressively destabilized by packing mutations of increasing
severity [3]. Any one of these variants can be used for sensing,
although for the typical case in which a balance between high Δθ
and low K d is desired, the mutant that yields outcome (i) is selected
as the final optimized product.
The AFF sensing mechanism is reversible and the speed at which
the forward and reverse conformational changes occur determines
how rapidly the sensor can detect fluctuations in analyte concentration. It may be desirable in some applications to increase this
response rate. For calbindin D 9k and RBP, the rate limiting step of
the N→N′ and N′→N reactions appears to involve a local but not
global unfolding event, most likely unfolding/dissociation of the
copy of the duplicate segment that is docked to the shared segment
of the protein. If this result is general, any mutation that destabilizes the interaction between the shared and duplicate regions has
the potential to accelerate the conformational switch. If one wishes
to attempt to engineer a faster AFF sensor, we recommend first
following all of the thermodynamic balancing procedures described
above, then introducing kinetic tuning mutation(s) in a manner
that does not perturb the existing N/N′ balance or ligand-binding
affinity. For example, consider a hypothetical 200-amino acid POI
from which residues 150–200 were duplicated to generate POI- AFF.
Further suppose that residues Phe130 and Leu170 pack tightly
against each other. Kinetic tuning could consist of a single
Phe130→Ala substitution in the shared region, or Leu170→Ala
+ Leu170′→Ala mutations in the respective duplicate segments.
In either case the N-fold and N′-fold are in principle destabilized
equally but the kinetic barrier to their interconversion may be
lowered, depending on the extent to which the Phe130-Leu170/
Leu170′ interaction is formed in the transition state ensemble.
As noted, it is simplest to fuse donor and acceptor FPs to the fulllength POI and duplicate fragment at the cloning stage; however, if
chemical fluorophores are desired then the labeling procedure is
identical to that of AFF. One difference of FREX is that the two components of the sensor are on average very far apart in the absence of
ligand (at reasonable protein concentrations), so donor/acceptor
pairs with a wide range of Förster distances can be considered.
5.3 Optional:
Kinetic Tuning
5.4 Modifications
to Step 3 for FREX
Engineering Allosteric Protein Switches