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Rationally improving LOV domain-based photoswitches.

Strickland D, Yao X, Gawlak G, Rosen MK, Gardner KH, Sosnick TR - Nat. Methods (2010)

Bottom Line: Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice.Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2).This approach can potentially be used to improve many AsLOV2-based photoswitches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

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Mutational stabilization LOV–Jα association and its effect on DNA binding activity in LovTAP. (a) Comparison of CD-derived xlitmut with NMR-derived xdarkmut for AsLOV2 with the indicated mutations. The identity line is shown as a reference. (b) Gel image of RsaI protection assay for LovTAP(G528A, N538E). Open and closed triangles denote reactant and product bands, respectively. (c) Effects of the helix-stabilizing mutations on LovTAP. In the upper panel, observed affinity constants (Kobs) are plotted against xeff. Large data points are average Kobs values, and small points show individual measurements. The data are compared to Eqs. 4 and 5 (solid and dashed lines). For wild-type LovTAP, xeff = xfus is calculated using Eq. 3. For mutants, xmut is determined by NMR, and xeff = xmutxfus. In the lower panel, β= Klitobs / Kdarkobs is plotted as a function of xeff and compared to Eq. 6. The shaded regions indicate regimes where β is relatively insensitive to changes in xeff.
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Figure 2: Mutational stabilization LOV–Jα association and its effect on DNA binding activity in LovTAP. (a) Comparison of CD-derived xlitmut with NMR-derived xdarkmut for AsLOV2 with the indicated mutations. The identity line is shown as a reference. (b) Gel image of RsaI protection assay for LovTAP(G528A, N538E). Open and closed triangles denote reactant and product bands, respectively. (c) Effects of the helix-stabilizing mutations on LovTAP. In the upper panel, observed affinity constants (Kobs) are plotted against xeff. Large data points are average Kobs values, and small points show individual measurements. The data are compared to Eqs. 4 and 5 (solid and dashed lines). For wild-type LovTAP, xeff = xfus is calculated using Eq. 3. For mutants, xmut is determined by NMR, and xeff = xmutxfus. In the lower panel, β= Klitobs / Kdarkobs is plotted as a function of xeff and compared to Eq. 6. The shaded regions indicate regimes where β is relatively insensitive to changes in xeff.

Mentions: Thus, for xeff < Kdarkhelix < Klithelix, β ≈ 1, i.e. nearly all of the effector is active, even in the dark state. Photoexcitation may further shift the equilibrium to the active state, e.g., from 99% active to 99.9% active, but this has a negligible functional effect. On the other hand, for xeff > Klithelix > Kdarkhelix, the maximum photoswitching dynamic range is obtained as β ≈ Klithelix / Kdarkhelix. For intermediate values of xeff, however, β increases rapidly with xeff, even assuming that xeff is the same in the lit and dark states (Fig. 1c and Fig. 2). Because β is small for LovTAP (5.3)2, we expect that the protein is near the lower end of this intermediate regime and we should be able to increase β with mutations that stabilize LOV–Jα docking.


Rationally improving LOV domain-based photoswitches.

Strickland D, Yao X, Gawlak G, Rosen MK, Gardner KH, Sosnick TR - Nat. Methods (2010)

Mutational stabilization LOV–Jα association and its effect on DNA binding activity in LovTAP. (a) Comparison of CD-derived xlitmut with NMR-derived xdarkmut for AsLOV2 with the indicated mutations. The identity line is shown as a reference. (b) Gel image of RsaI protection assay for LovTAP(G528A, N538E). Open and closed triangles denote reactant and product bands, respectively. (c) Effects of the helix-stabilizing mutations on LovTAP. In the upper panel, observed affinity constants (Kobs) are plotted against xeff. Large data points are average Kobs values, and small points show individual measurements. The data are compared to Eqs. 4 and 5 (solid and dashed lines). For wild-type LovTAP, xeff = xfus is calculated using Eq. 3. For mutants, xmut is determined by NMR, and xeff = xmutxfus. In the lower panel, β= Klitobs / Kdarkobs is plotted as a function of xeff and compared to Eq. 6. The shaded regions indicate regimes where β is relatively insensitive to changes in xeff.
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Figure 2: Mutational stabilization LOV–Jα association and its effect on DNA binding activity in LovTAP. (a) Comparison of CD-derived xlitmut with NMR-derived xdarkmut for AsLOV2 with the indicated mutations. The identity line is shown as a reference. (b) Gel image of RsaI protection assay for LovTAP(G528A, N538E). Open and closed triangles denote reactant and product bands, respectively. (c) Effects of the helix-stabilizing mutations on LovTAP. In the upper panel, observed affinity constants (Kobs) are plotted against xeff. Large data points are average Kobs values, and small points show individual measurements. The data are compared to Eqs. 4 and 5 (solid and dashed lines). For wild-type LovTAP, xeff = xfus is calculated using Eq. 3. For mutants, xmut is determined by NMR, and xeff = xmutxfus. In the lower panel, β= Klitobs / Kdarkobs is plotted as a function of xeff and compared to Eq. 6. The shaded regions indicate regimes where β is relatively insensitive to changes in xeff.
Mentions: Thus, for xeff < Kdarkhelix < Klithelix, β ≈ 1, i.e. nearly all of the effector is active, even in the dark state. Photoexcitation may further shift the equilibrium to the active state, e.g., from 99% active to 99.9% active, but this has a negligible functional effect. On the other hand, for xeff > Klithelix > Kdarkhelix, the maximum photoswitching dynamic range is obtained as β ≈ Klithelix / Kdarkhelix. For intermediate values of xeff, however, β increases rapidly with xeff, even assuming that xeff is the same in the lit and dark states (Fig. 1c and Fig. 2). Because β is small for LovTAP (5.3)2, we expect that the protein is near the lower end of this intermediate regime and we should be able to increase β with mutations that stabilize LOV–Jα docking.

Bottom Line: Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice.Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2).This approach can potentially be used to improve many AsLOV2-based photoswitches.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Genetically encoded protein photosensors are promising tools for engineering optical control of cellular behavior; we are only beginning to understand how to couple these light detectors to effectors of choice. Here we report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of Avena sativa phototropin 1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

Show MeSH