LIST OF ACRONYMS & ABBREVIATIONS

Transcription

1 LIST OF ACRONYMS & ABBREVIATIONS ALA ARG ASN ATD CRD CYS GLN GLU GLY GPCR HIS hstr ILE LEU LYS MET mglur1 NHDC PDB PHE PRO SER T1R2 T1R3 TMD TRP TYR THR 7-TM VFTM ZnSO 4 Alanine, A Arginine, R Asparagines, N Amino terminal domain cysteine rich domain Cysteine, C Glutamine, Q Glutamate, E Glycine, G G-protein coupled receptor Histidine, H Human sweet taste receptor Isoleucine, I Leucine, L Lysine, K Methionine, M Metabotropic Glutamate Receptor (Mouse) Neohesperidin dihydrochalcone Protein Data Bank Phenylalanine, F Proline, P Serine, S Taste receptor type 1 member 2 precursor Taste receptor type 1 member 3 precursor Transmembrane domain Tryptophan, W Tyrosine, Y Threonine 7 trans membrane helix Venus fly trap module Zinc Sulfate xi

2 LIST OF FIGURES Figure Caption Page 1.1 Plan of Study AH-B Glucophore model Ionization theory for taste response AH-B-X Glucophore model (Tripartite Glycophore Model) Multipoint Attachment Model for Glucophore Wedge shaped model Human sweet taste receptor structure Effect of metal ions on sweetness response of sweeteners Schematic Representation of Structure Prediction of hstr Representation of Identification of crucial residues and Glucophores Domain wise structure prediction: Templates and Tools used Schematic representation of methodology to identify crucial residues Optimized structures of sweet molecules Distribution of positively and negatively charged residues on sweet protein surfaces. Acidic residues are colored in blue and basic residues in red.a) brazzein, b) monellin, c) thaumatin, d) mabinlin, e) curculin and f) miraculin, 4.3 Distribution of Electrostatic potential shown in red and blue colour and hydrophobic patches in green colour on sweet protein surfaces. a) brazzein, b) monellin, c) thaumatin, d) mabinlin, e) curculin and f) miraculin Schematic representation of the plan of study Multiple sequence alignment result for a) T1R2 and b) T1R3 subunit of hstr Lengthwise representation of a) T1R2 and b) T1R3 structures 57 xii

3 predicted through different software 4.7 Predicted helix and Beta sheet in 3D structure of T1R2 subunit through various homology and threding based tools. Green color represents Beta sheet; Blue - Helix and orange lines are for loop regions 4.8 Predicted helix and Beta sheet in 3D structure of T1R3 subunit through various homology and threding based tools. Green color represents Beta sheet; Blue - Helix and orange lines are for loop regions 4.9 Predicted structures of -T1R2 domain receptor structure a) CPH model, b) Geno 3D, c) ITasser, d) 3 D Jigsaw, e) LOOP, f) HHPred, g) Phyre, h) Swiss Model, and T1R3 domain receptor structure i) CPH Model, j) Geno 3D, k) HHPred, l) ITasser m) 3D Jigsaw n) LOOP o) Phyre p) SwissModel TMD Domain receptor structure q) T1R2 ITasser, r) T1R3 ITasser, s) T1R3 Swiss GPCR + Prime Complete subunit receptor structure t) T1R2 ITasser, u) T1R3 ITasser, v) T1R2 Subunit (ATD-CRD from SWISS and TMD of ITasser) w) T1R3 subunit (ATD-CRD from CPH and TMD of Schrodinger) 4.10 Ramachandran plot of a) T1R2 and b) T1R3 complete subunit structure from ITASSSER; c) T1R2 built from SWISS-MODEL (ATD-CRD)+ I-TASSER (TMD) and d) T1R3 built from CPHmodels (ATD-CRD) + Swiss GPCR + Prime(TMD) 4.11 Validation of models of T1R2 and T1R3 predicted from different software a) Anolea and b) GROMOS (a) Validation of different models through Anolea (b) Validation of different models through Gromos Complex of (A) sodium cyclamate, Lactisole and NHDC with hstr in T1R3 TMD domain and (B) aspartame and Neotame in T1R2 ATD domain. Yellow lines represent H-bond interactions 4.14 Predicted models of human sweet taste receptor. (a-c) Three of the models with both transmembrane domain aligned alongside. d) One of the incorrect models with TMD of T1R2 aligned along ATD of T1R Docking of sweet proteins (red) with human sweet taste receptor (T1R2-T1R3 green-blue) a) brazzein, b) monellin, c) thaumatin d) mabinlin, e) curculin and f) miraculin Predicted active sites for T1R2 subunit Predicted active sites for T1R3 subunit 78 xiii

4 4.18 HEX docking results showing interaction of sweet molecules with receptor 4.19 LeadIT docking results showing interaction of sweet molecules with receptor Active sites of hstr with the docked molecules Schematic representation of Human Sweet Taste Receptor (hstr) and binding sites of sweet molecules 4.22 Effect of ZnSO 4 binding on AH-B-X distances of sweet molecules 4.23 Docked complex of sweet molecules with hstr in presence of metal ions 4.25 Effect of Point mutations on Hydrogen bond interactions between hstr and NHDC leading to unaltered sweetness response 4.26 Effect of Point mutations on Hydrogen bond interactions between hstr and NHDC leading to 5-6 fold decreased sweetness 4.27 Effect of Point mutations on Hydrogen bond interactions between hstr and NHDC leading to Eight fold decreased sweetness 4.28 Experimental data by Jiang & co-workers [Ref. 34] showing effect of Point mutations on Cyclamate sweetness response Glucophores for aspartame Glucophores for Sucralose Glucophores for Glucose Glucophores for Saccharin Glucophores for Sorbitol Glucophore for NHDC Glucophore for Cyclamate 134 xiv

5 LIST OF TABLES Table Caption Page 2.1 Frequently used sweetener molecules and their relative sweetness Adverse effects and Characteristics of Natural and Artificial Small 9 Sweet Molecules 2.3 Characteristics of Sweet proteins Characteristics of Sweet Taste Inhibitor Mutations on the receptor active sites Sweetness intensity of sweeteners and their chemical nature AH-B-X Glucophoretic distances of sweeteners List of sweet and non sweet molecules and their activity Comparison of Amino acid composition of sweet proteins and 49 their sweetness intensity 4.5 Topology of T1R2 and T1R3 as predicted from different topology 55 servers 4.6 Comparison of structure predicted by different homology and 56 threading based tools 4.7 Ramachandran Plot Analysis of structures predicted by different 62 software 4.8 Protein report of energy and steric clashes of models before and 67 after refinement 4.9 Predicted site of interaction of sweet molecules with T1R2 and 69 T1R3 subunits 4.10 Xtra precision docking results showing Glide score and energy of the ligand-receptor complex along with residues involved in hydrogen bond and hydrophobic interactions Interaction of Brazzein with hstr dimer models. Blue and red colored residues show T1R2 and T1R3 residues respectively. Yellow highlighted region is the domains of interaction of BRZhSTR. Highlighted residues are experimentally important residues 4.12 Interaction of sweet proteins with hstr dimer models. 74 Highlighted residues are experimentally 4.13 Active sites Residues of both T1R2 and T1R3 subunits Docking results from Hex Identified active site for sweet molecules using LEAD IT AutoDock docking results xv

6 4.17 Docking analysis of sweet molecules with hstr using Glide SP analysis of sweet molecules with hstr using Glide XP Hydrogen bond and hydrophobic interaction of sweet molecules 91 with hstr 4.20 Induced-fit docking analysis of sweet molecules interaction with 92 hstr 4.21 H- bond interactions involved in induced fit docking Hydrophobic interactions Active site identification of sweet molecules based on Induced fit 98 docking 4.24 AH-B-X glucophoretic distances before and after docking of sweet 99 molecules 4.25 Effect of ZnSO4 on AH-B-X glucophores Effect of metal ions on Hydrogen bond interactions Effect of metal ions on Hydrophobic interactions Crucial residues for sweet response Energy of NHDC-hSTR after mutation Hydrogen bond interactions of NHDC-hSTR after mutation Hydrophobic interactions between hstr NHDC Effect of Point mutations on Hydrogen bond interactions between 124 hstr and Cyclamate 4.33 Effect of Point mutations on Hydrophobic interactions between 126 hstr and Cyclamate 4.34 Glucophores for Aspartame Glucophores for Sucralose Glucophores for Glucose Glucophores for Saccharin Glucophores for Sorbitol 132 xvi