40
Thermodynamic tuning for FREX consists of binding experiments in which ligand is added to equimolar concentrations of
fluorescently tagged full-length POI and fragment. For example,
we optimized the Fn3-FREX sensor by screening the I75V, I75A,
and I75G tuning variants of the POI in the background of the
Y87A binding mutation (Fig. 2b). Fluorescence data are fit to Eq.
2. Our simulations have shown that it is possible, although highly
unlikely, that a ternary complex will form in the absence of a packing mutation, as the entropic barrier for intermolecular folding is
usually too large to be overcome by ligand-binding energy alone
[3]. A more realistic concern is that the binary complex of fulllength POI and fragment will form without ligand present. This
occurs when the full-length POI is “overbalanced,” i.e., so destabilized by the tuning mutation that restoration of the native packing interaction by the fragment drives folding in the absence of
ligand binding. It is not uncommon to span these extremes of stability by placing a set of packing mutations at a single well-chosen
site. A nicely balanced FREX construct is characterized by unusually
high Δθ (achieved by reducing FRET in the free state to near zero)
and K d similar to that of the WT POI. This condition was met in
the Fn3-FREX sensor by the I75A tuning mutation.
6 Concluding Remarks
Both AFF and FREX involve a folding competition akin to a twoperson game of molecular musical chairs: one copy of the duplicate
segment is left standing and is at least partially unfolded at any
given time. One must be alert to degradation and/or aggregation
originating from the orphaned copy. Computational methods for
predicting the stability and solubility of CPs, protein fragments,
and complexes thereof will greatly facilitate the design of FREX
and AFF sensors, as well as that of conformational switches based
on other mechanisms.
Acknowledgments
This work was supported by NIH grant R01 GM115762 to S.N.L.
References
1. Stratton MM, Mitrea DM, Loh SN (2008) A
Ca
2+
-sensing molecular switch based on alternate frame protein folding. ACS Chem Biol
3:723–732
2. Stratton MM, Loh SN (2010) On the mechanism of protein fold-switching by a molecular
sensor. Proteins 78:3260–3269
3. Zheng H, Bi J, Krendel M, Loh SN (2014)
Converting a binding protein into a biosensing
conformational switch using protein fragment
exchange. Biochemistry 53(34):5505–5514.
doi:10.1021/bi500758u
4. Ha JH, Shinsky SA, Loh SN (2013) Stepwise
conversion of a binding protein to a fluorescent
Jeung-Hoi Ha and Stewart N. Loh
Thermodynamic tuning for FREX consists of binding experiments in which ligand is added to equimolar concentrations of
fluorescently tagged full-length POI and fragment. For example,
we optimized the Fn3-FREX sensor by screening the I75V, I75A,
and I75G tuning variants of the POI in the background of the
Y87A binding mutation (Fig. 2b). Fluorescence data are fit to Eq.
2. Our simulations have shown that it is possible, although highly
unlikely, that a ternary complex will form in the absence of a packing mutation, as the entropic barrier for intermolecular folding is
usually too large to be overcome by ligand-binding energy alone
[3]. A more realistic concern is that the binary complex of fulllength POI and fragment will form without ligand present. This
occurs when the full-length POI is “overbalanced,” i.e., so destabilized by the tuning mutation that restoration of the native packing interaction by the fragment drives folding in the absence of
ligand binding. It is not uncommon to span these extremes of stability by placing a set of packing mutations at a single well-chosen
site. A nicely balanced FREX construct is characterized by unusually
high Δθ (achieved by reducing FRET in the free state to near zero)
and K d similar to that of the WT POI. This condition was met in
the Fn3-FREX sensor by the I75A tuning mutation.
6 Concluding Remarks
Both AFF and FREX involve a folding competition akin to a twoperson game of molecular musical chairs: one copy of the duplicate
segment is left standing and is at least partially unfolded at any
given time. One must be alert to degradation and/or aggregation
originating from the orphaned copy. Computational methods for
predicting the stability and solubility of CPs, protein fragments,
and complexes thereof will greatly facilitate the design of FREX
and AFF sensors, as well as that of conformational switches based
on other mechanisms.
Acknowledgments
This work was supported by NIH grant R01 GM115762 to S.N.L.
References
1. Stratton MM, Mitrea DM, Loh SN (2008) A
Ca
2+
-sensing molecular switch based on alternate frame protein folding. ACS Chem Biol
3:723–732
2. Stratton MM, Loh SN (2010) On the mechanism of protein fold-switching by a molecular
sensor. Proteins 78:3260–3269
3. Zheng H, Bi J, Krendel M, Loh SN (2014)
Converting a binding protein into a biosensing
conformational switch using protein fragment
exchange. Biochemistry 53(34):5505–5514.
doi:10.1021/bi500758u
4. Ha JH, Shinsky SA, Loh SN (2013) Stepwise
conversion of a binding protein to a fluorescent
Jeung-Hoi Ha and Stewart N. Loh