Limits...
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.

Show MeSH
Conformational and binding equilibria in AsLOV2 and LovTAP. (a) Photoexcitation (hν) of AsLOV2 (blue) is accompanied by displacement and unfolding of the Jα helix (dark blue). (b) In AsLOV2 (left), photoactivation shifts the equilibrium from mostly docked to mostly undocked. Black lines, dark-state free energy surfaces; dashed lines, lit-state free energy surfaces. Upon fusion with an effector (center), the equilibrium is shifted due to interactions with the effector and the helix is mostly undocked in the dark and lit states. As a consequence, the effector is mostly active in both states. Helix stabilizing mutations specifically stabilize the docked state (ΔG = – RT ln xeff), bringing the equilibrium back into a regime where the effector is mostly inactive in the dark, and photoactivation shifts the equilibrium from mostly inactive to mostly active. (c) Khelix, equlibrium constant of helix undocking for the isolated LOV2 domain. The fusion protein LovTAP is in equilibrium between an inactive conformation (left), in which the shared helix is docked on the LOV domain, and an active conformation (center), in which it is docked on the TrpR domain. Due to competition by TrpR (orange) for the helix, the equilibrium constant of the helix undocking reaction, Khelix, is increased by a factor 1 / xeff. In the active conformation, LovTAP binds DNA with an intrinsic association constant KDNA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2914111&req=5

Figure 1: Conformational and binding equilibria in AsLOV2 and LovTAP. (a) Photoexcitation (hν) of AsLOV2 (blue) is accompanied by displacement and unfolding of the Jα helix (dark blue). (b) In AsLOV2 (left), photoactivation shifts the equilibrium from mostly docked to mostly undocked. Black lines, dark-state free energy surfaces; dashed lines, lit-state free energy surfaces. Upon fusion with an effector (center), the equilibrium is shifted due to interactions with the effector and the helix is mostly undocked in the dark and lit states. As a consequence, the effector is mostly active in both states. Helix stabilizing mutations specifically stabilize the docked state (ΔG = – RT ln xeff), bringing the equilibrium back into a regime where the effector is mostly inactive in the dark, and photoactivation shifts the equilibrium from mostly inactive to mostly active. (c) Khelix, equlibrium constant of helix undocking for the isolated LOV2 domain. The fusion protein LovTAP is in equilibrium between an inactive conformation (left), in which the shared helix is docked on the LOV domain, and an active conformation (center), in which it is docked on the TrpR domain. Due to competition by TrpR (orange) for the helix, the equilibrium constant of the helix undocking reaction, Khelix, is increased by a factor 1 / xeff. In the active conformation, LovTAP binds DNA with an intrinsic association constant KDNA.

Mentions: Our understanding of the light-regulated undocking of the Jα helix in AsLOV2 has paved the way for its use as the input component of designed proteins2,3,5. In each of these three engineered proteins, effector activity is suppressed in the dark, concomittant with the Jα helix being in the folded and docked conformation. Although the Jα is mostly docked in the dark state and mostly undocked in the lit state, both conformations are populated in both the dark and lit states (Fig. 1a). The equilibrium between these two populations dramatically shifts between the dark and lit states, providing a large thermodynamic driving force (ΔG ≈ 4 kcal/mol) which can be used to regulate the activity of an effector17.


Rationally improving LOV domain-based photoswitches.

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

Conformational and binding equilibria in AsLOV2 and LovTAP. (a) Photoexcitation (hν) of AsLOV2 (blue) is accompanied by displacement and unfolding of the Jα helix (dark blue). (b) In AsLOV2 (left), photoactivation shifts the equilibrium from mostly docked to mostly undocked. Black lines, dark-state free energy surfaces; dashed lines, lit-state free energy surfaces. Upon fusion with an effector (center), the equilibrium is shifted due to interactions with the effector and the helix is mostly undocked in the dark and lit states. As a consequence, the effector is mostly active in both states. Helix stabilizing mutations specifically stabilize the docked state (ΔG = – RT ln xeff), bringing the equilibrium back into a regime where the effector is mostly inactive in the dark, and photoactivation shifts the equilibrium from mostly inactive to mostly active. (c) Khelix, equlibrium constant of helix undocking for the isolated LOV2 domain. The fusion protein LovTAP is in equilibrium between an inactive conformation (left), in which the shared helix is docked on the LOV domain, and an active conformation (center), in which it is docked on the TrpR domain. Due to competition by TrpR (orange) for the helix, the equilibrium constant of the helix undocking reaction, Khelix, is increased by a factor 1 / xeff. In the active conformation, LovTAP binds DNA with an intrinsic association constant KDNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2914111&req=5

Figure 1: Conformational and binding equilibria in AsLOV2 and LovTAP. (a) Photoexcitation (hν) of AsLOV2 (blue) is accompanied by displacement and unfolding of the Jα helix (dark blue). (b) In AsLOV2 (left), photoactivation shifts the equilibrium from mostly docked to mostly undocked. Black lines, dark-state free energy surfaces; dashed lines, lit-state free energy surfaces. Upon fusion with an effector (center), the equilibrium is shifted due to interactions with the effector and the helix is mostly undocked in the dark and lit states. As a consequence, the effector is mostly active in both states. Helix stabilizing mutations specifically stabilize the docked state (ΔG = – RT ln xeff), bringing the equilibrium back into a regime where the effector is mostly inactive in the dark, and photoactivation shifts the equilibrium from mostly inactive to mostly active. (c) Khelix, equlibrium constant of helix undocking for the isolated LOV2 domain. The fusion protein LovTAP is in equilibrium between an inactive conformation (left), in which the shared helix is docked on the LOV domain, and an active conformation (center), in which it is docked on the TrpR domain. Due to competition by TrpR (orange) for the helix, the equilibrium constant of the helix undocking reaction, Khelix, is increased by a factor 1 / xeff. In the active conformation, LovTAP binds DNA with an intrinsic association constant KDNA.
Mentions: Our understanding of the light-regulated undocking of the Jα helix in AsLOV2 has paved the way for its use as the input component of designed proteins2,3,5. In each of these three engineered proteins, effector activity is suppressed in the dark, concomittant with the Jα helix being in the folded and docked conformation. Although the Jα is mostly docked in the dark state and mostly undocked in the lit state, both conformations are populated in both the dark and lit states (Fig. 1a). The equilibrium between these two populations dramatically shifts between the dark and lit states, providing a large thermodynamic driving force (ΔG ≈ 4 kcal/mol) which can be used to regulate the activity of an effector17.

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