Modulating protein activity using tethered ligands with mutually exclusive binding sites

The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.

Error! Reference source not found., which we consider as a good estimate for the CLASH constructs.
In the absence of effector molecule, the tethered ligand should bind intramolecularly to its cognate receptor. If we stipulate that the CLASH construct should be to more than 90% in its closed conformation, we can calculate the minimal dissociation constant for the tethered ligand (K d1 ) for the reporter protein as at most one tenth of the effective molarity, and thus around 10 µM. The amount of the effector protein to obtain half of the activation of the reporter depends on the K d2 for its respective ligand used in the CLASH, and can be estimated from the formula M eff K d2 /K d1 . For example if K d1 is 10 µM, a K d2 of 10 nM results in half-activation of the reporter at 100 nM effector concentration. The kinetics of the CLASH constructs is mainly governed by the unbinding of the intramolecular ligand from the respective receptor protein, while closing is driven by the unbinding of the effector protein, as was previously described for the similar semisynthetic biosensors SNIFITs [3]. In case of processes involving very slow off-rates, as for examples for the unbinding of biotin from streptavidin, the activation after the addition of the protein can be considered practically irreversible. On the contrary, in Figure 4 we demonstrated that CLASH-AChE/HCA is reversibly changing its conformation upon perfusion and removal of the effector tacrine. In this case the transition kinetics upon addition of tacrine can be estimated by the k off of the tethered edrophonium from AChE; while upon removal of tacrine two mechanisms need to be considered: unbinding of the intramolecular sulfonamide from HCA and unbinding of tacrine from AChE; since the latter is the slower, the kinetic of the transition is governed by the k off of tacrine from AChE.

SUPPLEMENTARY METHODS: Synthetic procedures
All reactions were carried out in oven-dried glassware under nitrogen atmosphere, unless stated otherwise.  temperature. In a standard coupling reaction, the carboxylic acid, the coupling agent (TSTU or HBTU) and the amine to be coupled were dissolved in separate flasks in anhydrous DMSO to a final concentration of 10 to 500 mM. 1 eq of the carboxylic acid solution was treated with 2.5 eq DIEA, followed by 1.2 eq of the coupling agent solution and stirred at room temperature. Activated ester formation was checked by TLC or analytical HPLC and, when complete, 1-3 eq of the amine solution was added. The reaction was further stirred at room temperature until the activated ester was completely consumed. The reaction was then quenched by addition of water and acidified with 3 eq acetic acid, before Prep-HPLC purification.
General Procedure 2 -Phenol alkylation A typical reaction for alkylating a phenolic group was performed dissolving 1 eq of phenol in anhydrous DMF to a final concentration of 10-100 mM, followed by 1 eq of the alkyl electrophile, 2 eq of anhydrous potassium carbonate and an excess of MS4A. The suspension was vigorously stirred at 55C until the reaction was judged complete by analytical-HPLC or TLC.

General Procedure 3 -Boc deprotection
The tert-butylcarbamate-protected compound was dissolved in an excess of TFA and stirred at room temperature.
When the protecting group was completely removed as judged by analytical-HPLC, the excess of solvent and side products were removed at reduced pressure.

General Procedure 4-Fmoc deprotection
The 9-Fluorenylmethylcarbamate -protected compound was dissolved in acetonitrile and 2.5 eq of DBU were added. The solution was stirred until analytical HPLC indicated complete protecting group removal, and then acidified with 3 eq of glacial acetic acid. The solution was concentrated at reduced pressure and purified by preparative HPLC.
General Procedure 5 -Cy3 derivatives preparation Cy3 derivative synthesis was performed using a modification of General Procedure 1 using TSTU as coupling agent in a 2-step one-pot reaction, starting from the previously described bis-carboxy-Cy3 derivative 23 30        The synthesis of the edrophonium derivative 27 was previously described [2]. Compound 27 and was coupled to Compound 17 according to General Procedure 1. The crude