Fabrication of molecular tension probes

A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein–protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for determining molecular tension appended by ligand-activated PPIs. The summary of the method is: • Molecular tension appended by protein–protein interactions (PPI) is visualized with a luciferase.• Estrogen activities are quantitatively illuminated with the molecular tension probes.• Full-length Renilla luciferase enhances the optical intensities after bending by PPI.

We previously developed a unique bioluminescent probe called "strain probe" for illuminating PPIs [7]. We initially hypothesized that any luciferase has talent to change its enzymatic activity according to the molecular tension artificially appended by PPIs. This molecular tension may cause distortion of the active site, which modulates the enzymatic activity.
In this method, we introduce how to fabricate molecular tension probes emitting bioluminescence in response to molecular tension appended by PPIs in detail. Basic concept for designing molecular tension probes

Materials
The basic design of molecular tension probes consists of four different ingredients, i.e., a full-length luciferase, a pair of proteins of interest (called proteins "A" and "B"), and a flexible linker, where the luciferase is sandwiched between the two proteins of interest via a minimal length of flexible linkers (Fig. 1). The luciferase is tensed by the ligand-activated PPIs. The minimal flexible linkers as possible connecting the ingredients are advantageous to efficiently convert the molecular tension to practical distortion of the sandwiched luciferase.
Any luciferases basically have talent to vary their enzymatic activity more or less according to the molecular tension appended by an intra-molecular PPI. A globular marine luciferase may be advantageous over beetle luciferases, which consist of N-and C-terminal domains connected by a flexible hinge region [8] (Fig. 1A). A globular marine luciferase like Renilla reniformis luciferase (RLuc) easily receives tension from PPIs, whereas the flexible region in beetle luciferases relaxes the intramolecular tension. 1 The active site of RLuc8 is close from the C-terminal end [10], thus is prone to be influenced by protein-tagging and molecular tension appended by adjacent proteins.
In this protocol, we exemplify a molecular tension probe that is made of RLuc8 as a model luciferase sandwiched between the ligand-binding domain of the human estrogen receptor (ER LBD) as an intracellular receptor member of the nuclear receptor superfamily and Src homology domain 2 of n-Src (SH2), based on our previous papers [7,11]. Upon ligand activation, Tyr537 of ER LBD is 1 The C-terminal domain of firefly luciferase (FLuc) is rotated to release luciferin in the light-emitting process [9], which is hampered by the molecular tension in the probe. phosphorylated and recognized by the counterpart SH2 domain. This molecular tension varies the bioluminescence intensity of the sandwiched RLuc8 (Fig. 1). The corresponding positive and negative control studies in a tension-free condition were well discussed in the original paper [7].

Preparation of the cDNA constructs encoding a molecular tension probe
The cDNA constructs encoding molecular tension probes is fabricated by conventional genetic engineering techniques including polymerase chain reaction (PCR) with an adequate primer set and its subcloning into a mammalian expression vector as follow (Fig. 1). Procedure 1. Generate the cDNA segments encoding full-length Renilla luciferase 8 (1-311 aa; RLuc8) by PCR using a corresponding primer set flanked with unique restriction sites, BamHI and KpnI, for introducing unique restriction sites at the 5 0 -and 3 0 -terminals, respectively. 2. Fabricate the cDNA segments encoding the ER LBD and the SH2 domain of v-Src by PCR using corresponding primer sets flanked with the unique restriction sites, HindIII/BamHI, and KpnI/XhoI, respectively. 3. Digest the above cDNA segments and the multiple cloning site (MCS) of a mammalian expression vector pcDNA 3.1(+) (Invitrogen) with the corresponding restriction enzymes, HindIII/BamHI; BamHI/KpnI; KpnI/XhoI; HindIII/KpnI, respectively (Fig. 1B). 2 4. Gel-purify the digested cDNA segments and the mammalian expression vector.

Bioluminescence spectra of COS-7 cells carrying pErs
The ligand-driven variance of optical spectra can be determined with a mammalian culture cell line, COS-7 cells, expressing the molecular tension probe as follows ( Fig. 2A).

Grow COS-7 cells derived from African green monkey kidney fibroblast 4 in a Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% penicillin/streptomycin (P/S; Gibco) at 37 C in a 5% CO 2 incubator.   [7]. (B) Ligand selectivity of ERS. The luminescence intensities were compared after activation of ERS with various ligands. The figure was modified from our previous study [7]. (C) An optical image of the lysates of COS-7 cells after stimulation with vehicle or OHT (n = 9). 3 In this protocol, the made probes are named "ERS" from the consecutive initial letters of the components, i.e., ER LBD-RLuc8-SH2. The corresponding plasmid was named pErs. 4 Any mammalian cell lines are basically applicable for this protocol. 5 The incubation time influences on the signal-to-background (S/B) ratios. A better S/B ratio is achieved by avoiding the overexpression. 6 Recommended is a spectra photometer equipped with a cooled CCD camera which simultaneously acquires the whole range of wavelength, instead of scanning the wavelength range.
Determination of estrogenic activity of chemicals with a molecular tension probe Estrogenicity of steroidal hormones and synthetic chemicals is visualized with a molecular tension probe, ERS, as follows (Fig. 2B). where the RLU is an amplified value of photon counts generated from the luminometer (arbitrary unit) (Fig. 2B).
The procedure from Steps 5 to 7 may be substituted by the following alternative (Fig. 2C).
5a. Transfer an aliquot of the cell lysates (10 mL) to each well of a fresh 96-well optical bottom plate. 8 6a. Simultaneously inject an aliquot (50 mL) of the assay buffer dissolving nCTZ into the cell lysates on the plate using a multichannel pipette 9 and immediately transfer the plate in an image analyzer (LAS-4000, FujiFilm). 7a. Determine the optical intensities in the plate with the equipped controlling software (Image reader ver2.0) and analyze the optical images with the analysis software (Multi Gauge ver3.1).
Bioluminescence imaging (BLI) of living mammalian cells with a molecular tension probe Live-cell images by the tension probe are conducted with the following procedure (Fig. 3).