Supplemental Data
Files in this Data Supplement:
- Supplemental Figures 1-6 (.pdf, 947 KB) - Supp. Fig. 1. Multiple protein sequence alignment of 188 T2Rs derived from the NCBI database aligned using Clustal v2.1. Supp. Fig. 2. Immunofluorescence microscopy showing localization of WT and mutant T2R1 constructs transiently expressed in HEK293T cells. The WT and mutant T2R1 receptor expression was visualized using goat anti-mouse Alexafluor 488 secondary antibody (panel A, green) and the ER was visualized with goat anti-rabbit Alexafluor 594 secondary antibody (panel B, red). The nucleus stained with Hoechst-33342 dye is shown in blue (panel C). The overlay of the receptor expression (indicated by white arrows), ER and nucleus staining is shown in panel D. Supp. Fig. 3. Molecular models of wild type T2R1, N24D, I27A and I27V mutants. In wild type, Iso27(1.53) makes multiple backbone-backbone interactions with Asn24(1.50), Thr23(1.49) and with Asn31 on the cytoplasmic side. The N24D mutant loses backbone contacts with Iso27 and Val28, while its side chain carboxyl oxygen forms a new bond with Gln59(2.58) on TM2. These interactions cause relaxation in the helical backbone around the residue 1.50 in the N24D mutant. In the I27A mutant, Ala27 loses the backbone contact with Asn24(1.50), and a key interaction involving Arg55(2.54) with Asn24(1.50) was also lost. Interestingly, this H-bonding involving Arg55(2.54) with Asn241.50 was present in the I27V mutant. The interhelical hydrogen bond between Asn24(1.50) and Arg55(2.54) restrains receptor activity, as loss of this bond in I27A mutant results in hyperactive receptor. Supp. Fig. 4. Molecular models of wild type T2R1, R55A, R55K, H273S and H273F mutants. Interactions of conserved amino acids Asn24(1.50), Iso27(1.53), Arg55(2.54) and His273(7.46) in wild type T2R1. Both the R55A and R55K mutants lose the H-bonding with Asn24(1.50) and Ser274(7.47), while the side chain of Lys55 mutant is restrained with new H-bonding contacts with side chain functional group of Glu92 and backbone carbonyl of Leu51. The interhelical hydrogen bond between Asn24(1.50) and Arg55(2.54) restrains receptor activity, as loss of this bond in R55A mutant results in hyperactive receptor. His273 might play an indirect role in maintaining TM1-TM2-TM7 contacts, for example, the restraining interaction of Arg55(2.54) with Asn24(1.50) was lost in the H273F mutant. Supp. Fig. 5. Molecular models of N66D and E74A mutants, residues important for agonist DXM binding in T2R1. Supp. Fig. 6. Molecular model of T2R1 showing the LxxSL motif on the cytoplasmic end of TM5. The network of intrahelical H-bond interactions between backbones of the three residues, Leu197(5.46), Ser200(5.49), and Leu201(5.50) are shown in the wild type (WT). The L197V, S200T, and L201V mutations cause a significant loss of these interactions resulting in protein misfolding and/or non-functional receptor. In case of S200T mutant, an additional interaction with the backbone of His204 was lost. The His204 of intracellular loop 3 (ICL3) is highly conserved (96%) across T2Rs.
- Supplemental Table 1 (.doc, 25 KB) - Taste sensory analysis of bitter compounds using E-tongue.
- Supplemental Table 2. (.doc, 37 KB) - Summary of activation profile and immunofluorescence expression of highly conserved and T2R1 specific transmembrane mutant receptors.