Part:BBa_K2368003:Experience

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Applications of BBa_K2368003

In our experiment, this part is used to accept the signal inputted in our whole pathway.

Part 1: Dry experiments of T1R2

1、Structure models

Purpose

In order to confirm whether this “radar” T1R2/T1R3 can “sense” the sweetness of different sweetener, we simulate the receptor structure model. It helps us to understand how the sweeteners binding to T1R2/T1R3 receptor visually. Otherwise we hope to find some unknown sweeteners binding sites based on this model, even some ideal but unknown sweeteners.

Method

Initially, to make the signal input more accurate and reliable, we simulate the T1R2/T1R3 receptor structure model using SWISS-MODEL. Additionally, we prepare some sweeteners’ PDB file based on Chemdraw 2D and Chemdraw 3D. And the docking process is performed by using Autodock Vina.

Result

We used homology modeling method to get the structure of human sweet receptorT1R2/T1R3. The quality of the simulate structure within normal range and it only contain the ligand-binding-domain based on the crystal protein structure of Mice.

Fig. 2 The simulate structure of human sweetness receptors ligand-binding domain (LBD)

Then we use software Chemdraw 2D and Chemdraw 3D to build PDB files of some classic sweeteners.

Fig. 3 The 3D structure of some sweeteners

The docking process is carried out under Autodock Vina (Fig. 4-6).

Fig. 4 The docking result of different sweeteners

Fig. 5 The docking result of aspartame

Fig. 6 The docking result of stevioside

2、T1R2/T1R3 receptor activation part

The part is about the sweeteners binding to the receptor and the GPCR produce the initial signal through changing structure. For T1R2/T1R3, we divide the process of inducing signal into four conditions of T1R2/T1R3 as show in Fig. 7. And the reaction between different conditions of protein is used ODEs to describe it. The all ODEs are shown follow.

Fig. 7 T1R2/T1R3 receptor activation reaction diagram

(T1R2/3: the T1R2/T1R3 heterodimer)

The parameters of this part are listed in the Table 1.

The output of the T1R2/T1R3 receptor activation part is showed. (Fig.8)

Fig. 8 The output signal, activated state of T1R2/T1R3, induced by different concentration ligand

As we can see, the activated state of T1R2/T1R3 establishes clear distant under different concentration of ligand for binding. It can prove that this pathway can sense different strength of signal and the signal will decrease soon, which means our system will have less signal interfere.

3、The result of T1R2 expression

As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which will is similar to our experimental data. Finally, the RFP of all groups reduce slowly.

Fig. 9 The simulation result of RFP intensity in population level.

Part 2: Wet experiments of T1R2

Design:

According to data mining, we synthesized the T1R2 gene by OE PCR. we fused myc, his tag at the N-terminus of sweetness receptor to detect whether it localize the cell membrane. And they are ligated to the plasmid by Gibson assembly.

Fig. 10 The schematic diagram of expressing T1R2.

Result

In our experiment, the T1R2 gene is used to accept the signal inputted in our whole pathway. We synthesized this part by OE PCR.

Fig.11 The electrophoresis of the positive result of T1R2.

Then the expression and location of the receptor are detected by immunofluorescence. The principle of immunofluorescence is that the antibody A binds to the tag, and then the fluorescent antibody B binds to the A. This is the positive result of our immunofluorescence.

Fig.12 Immunofluorescence of T1R2/T1R3 in yeast. The scale bars represent 10µm

As the result of combination model established, we can see the curve of RFP intensity does not increase at first 5 hours, and then the RFP intensity increases rapidly among 5-15 hours. At the same time, the differences of each ligand concentration are displayed in the middle of culture time (around at 15 hours), which will is similar to our experimental data. Finally, the RFP of all groups reduce slowly after 15 hours.

Fig. 13 The fluorescence intensity of different Sweeteners

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[3] Bohlmann L, Tredwell G D, Yu X, et al. Nature Chemical Biology, 2015, 11(12):955.

[4] Dubois G E. Molecular mechanism of sweetness sensation.[J]. Physiology & Behavior, 2016, 164(Pt B):453.

[5] Kofahl B, Klipp E. Modelling the dynamics of the yeast pheromone pathway.[J]. Yeast, 2004, 21(10):831.

[6] Richardson, Kathryn. Mechanisms of GPCR signal regulation in fission yeast[J]. University of Warwick, 2014.

[7] Nie Y, Vigues S, Hobbs J R, et al. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli.[J]. Current Biology Cb, 2005, 15(21):1948-52.

[8] Audet M, Bouvier M. Restructuring G-Protein- Coupled Receptor Activation[J]. Cell, 2012, 151(1):14-23.

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