data_18440 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; Analog of the fragment 197-221 of 1- adrenoreceptor ; _BMRB_accession_number 18440 _BMRB_flat_file_name bmr18440.str _Entry_type original _Submission_date 2012-05-04 _Accession_date 2012-05-04 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details 'Analog of the fragment 197-221 of 1- adrenoreceptor' loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Bushuev Valery N. . 2 Zykov K. A. . 3 Bespalova Z. D. . 4 Bibilashvili R. Sh. . 5 Efremov E. E. . 6 Golitsyn S. P. . 7 Kaznacheeva L. I. . 8 Kuzhetsova T. V. . 9 Levashov P. S. . 10 Masenko V. P. . 11 Mironova N. A. . 12 Molokoedov F. S. . 13 Rutkevich P. N. . 14 Rvacheva A. V. . 15 Sharf T. V. . 16 Sidorova M. V. . 17 Vlasik T. N. . 18 Yanushevskaya E. V. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 139 "13C chemical shifts" 63 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2013-05-03 original author . stop_ _Original_release_date 2013-05-03 save_ ############################# # Citation for this entry # ############################# save_citations _Saveframe_category entry_citation _Citation_full . _Citation_title 'Not applicable' _Citation_status 'in preparation' _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID ? loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Bushuev Valery N. . stop_ _Journal_abbreviation 'To be Published' _Journal_volume . _Journal_issue . _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first . _Page_last . _Year . _Details . save_ ################################## # Molecular system description # ################################## save_assembly _Saveframe_category molecular_system _Mol_system_name 'Analog of the fragment 197-221 of 1- adrenoreceptor' _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label 'Analog of the fragment 197-221 of 1- adrenoreceptor' $entity stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state ? _System_paramagnetic no _System_thiol_state . _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_entity _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common entity _Molecular_mass 2941.249 _Mol_thiol_state 'all disulfide bound' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 25 _Mol_residue_sequence ; NCWRAESDEARRCYNDPKCS DSVCK ; loop_ _Residue_seq_code _Residue_label 1 ASN 2 CYS 3 TRP 4 ARG 5 ALA 6 GLU 7 SER 8 ASP 9 GLU 10 ALA 11 ARG 12 ARG 13 CYS 14 TYR 15 ASN 16 ASP 17 PRO 18 LYS 19 CYS 20 SER 21 ASP 22 SER 23 VAL 24 CYS 25 LYS stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date 2014-05-12 loop_ _Database_name _Database_accession_code _Database_entry_mol_name _Sequence_query_to_submitted_percentage _Sequence_subject_length _Sequence_identity _Sequence_positive _Sequence_homology_expectation_value PDB 2LSQ "Analog Of The Fragment 197-221 Of Beta-1 Adrenoreceptor" 100.00 25 100.00 100.00 3.67e-08 stop_ save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $entity Human 9606 Eukaryota Metazoa Homo sapiens stop_ save_ ######################### # Experimental source # ######################### save_experimental_source _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_type _Vector_name _Details $entity 'chemical synthesis' . none none . none none 'The peptide was prepared by automatic solid phase synthesis (SPPS) using the Fmoc strategy starting from 0.25 mmol of commercial N Fmoc-Lys(Boc)-polymer (Bachem, Switzerland). A copolymer of styrene with 1% divinylbenzene with a hydroxymethylphenyloxymethyl anchoring group (the Wang-polymer) was used as a carrier. The side functions of the amino acids were blocked by the following protective groups: But for hydroxyl functions of Ser and Tyr; But for carboxyl functions of Asp and Glu; Boc for -amino functions of Lys; Trt for carboxylamide group of Asn, sulfhydryl functions of residues Cys198 and Cys220; Acm for sulfhydryl functions of residues Cys209 and Cys215, and Pbf for guanidine function of Arg. The amino acid chain was elongated stepwise starting from C-terminus using carbodiimide in the presence of 1-hydroxybenzotriasole. When the SPPS was finished, the peptide was cleaved from carrier with the simultaneous removal of the acid-labile protective groups by treatment with trifluoracetic acid with the addition of water, triisopropylsilane and dithiothreitole. The synthesized [Cys(Acm)2]peptide was purified by HPLC to a purity 85-90%. The intramolecular S-S bridge between Cys198 and Cys220 was with hydrogen peroxide at pH 8. The target monocyclic intermediate of peptide was purified by HPLC to a purity 90-95%. The product was characterized by MALDI-TOF mass spectrometry. The second disulfide bond between Cys209 and Cys215 was formed by the treatment of monocyclic intermediate [Cys(Acm)2]peptide with iodine (for simultaneous cleavage of Acm group and formation S-S bridge) in methanol. The bicyclic target product was purified by HPLC to a purity 98% and characterized by MALDI-TOF mass spectrometry.' stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_sample_1 _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $entity 2 mM 'natural abundance' 'sodium azide' 1 mM 'natural abundance' 'sodium acetate' 20 mM '[U-100% 2H]' H2O 90 % 'natural abundance' D2O 10 % 'natural abundance' stop_ save_ save_sample_2 _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $entity 2 mM 'natural abundance' 'sodium azide' 1 mM 'natural abundance' 'sodium acetate' 20 mM '[U-100% 2H]' D2O 100 % 'natural abundance' stop_ save_ ############################ # Computer software used # ############################ save_xwinnmr _Saveframe_category software _Name xwinnmr _Version 1.3 loop_ _Vendor _Address _Electronic_address 'Bruker Biospin' . . stop_ loop_ _Task collection processing stop_ _Details . save_ save_CARA _Saveframe_category software _Name CARA _Version 1.9.5 loop_ _Vendor _Address _Electronic_address 'Keller and Wuthrich' . . stop_ loop_ _Task 'chemical shift assignment' 'peak picking' stop_ _Details . save_ save_CYANA _Saveframe_category software _Name CYANA _Version 3.0 loop_ _Vendor _Address _Electronic_address 'Guntert, Mumenthaler and Wuthrich' . . stop_ loop_ _Task 'structure solution' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_spectrometer_1 _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model DPX _Field_strength 500 _Details . save_ ############################# # NMR applied experiments # ############################# save_2D_1H-13C_HSQC_aliphatic_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-13C HSQC aliphatic' _Sample_label $sample_2 save_ save_2D_1H-13C_HSQC_aromatic_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-13C HSQC aromatic' _Sample_label $sample_2 save_ save_2D_DQF-COSY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '2D DQF-COSY' _Sample_label $sample_2 save_ save_2D_1H-1H_NOESY_4 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H NOESY' _Sample_label $sample_2 save_ save_2D_1H-1H_TOCSY_5 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H TOCSY' _Sample_label $sample_2 save_ save_2D_1H-1H_NOESY_6 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H NOESY' _Sample_label $sample_1 save_ save_2D_1H-1H_TOCSY_7 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H TOCSY' _Sample_label $sample_1 save_ ####################### # Sample conditions # ####################### save_sample_conditions_1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 21 . mM pH 5.5 . pH pressure 1 . atm temperature 303 . K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chemical_shift_reference_1 _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio DSS C 13 'methyl protons' ppm 0.00 na indirect . . . 0.251449530 DSS H 1 'methyl protons' ppm 0.00 internal direct . . . 1.000000000 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_assigned_chem_shift_list_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Experiment_label '2D 1H-13C HSQC aliphatic' '2D 1H-13C HSQC aromatic' '2D 1H-1H NOESY' '2D 1H-1H TOCSY' stop_ loop_ _Sample_label $sample_2 $sample_1 stop_ _Sample_conditions_label $sample_conditions_1 _Chem_shift_reference_set_label $chemical_shift_reference_1 _Mol_system_component_name 'Analog of the fragment 197-221 of 1- adrenoreceptor' _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 2 2 CYS HA H 4.773 0.005 1 2 2 2 CYS HB2 H 2.993 0.005 2 3 2 2 CYS HB3 H 2.936 0.005 2 4 2 2 CYS CB C 40.326 0.100 1 5 3 3 TRP H H 8.320 0.005 1 6 3 3 TRP HA H 4.946 0.005 1 7 3 3 TRP HB2 H 3.222 0.005 2 8 3 3 TRP HB3 H 2.999 0.005 2 9 3 3 TRP HD1 H 7.064 0.005 1 10 3 3 TRP HE1 H 9.961 0.005 1 11 3 3 TRP HE3 H 7.471 0.005 1 12 3 3 TRP HZ2 H 7.425 0.005 1 13 3 3 TRP HZ3 H 7.056 0.005 1 14 3 3 TRP HH2 H 7.173 0.005 1 15 3 3 TRP CA C 52.486 0.100 1 16 3 3 TRP CB C 28.198 0.100 1 17 4 4 ARG H H 8.730 0.005 1 18 4 4 ARG HA H 4.244 0.005 1 19 4 4 ARG HB2 H 1.883 0.005 2 20 4 4 ARG HB3 H 1.712 0.005 2 21 4 4 ARG HG2 H 1.669 0.005 2 22 4 4 ARG HG3 H 1.604 0.005 2 23 4 4 ARG HD2 H 3.188 0.005 1 24 4 4 ARG HD3 H 3.188 0.005 1 25 4 4 ARG HE H 7.308 0.005 1 26 4 4 ARG CA C 54.012 0.100 1 27 4 4 ARG CB C 27.947 0.100 1 28 4 4 ARG CG C 24.815 0.100 1 29 4 4 ARG CD C 40.618 0.100 1 30 5 5 ALA H H 8.787 0.005 1 31 5 5 ALA HA H 4.060 0.005 1 32 5 5 ALA HB H 1.398 0.005 1 33 5 5 ALA CA C 51.375 0.100 1 34 5 5 ALA CB C 16.150 0.100 1 35 6 6 GLU H H 8.728 0.005 1 36 6 6 GLU HA H 4.156 0.005 1 37 6 6 GLU HB2 H 2.133 0.005 2 38 6 6 GLU HB3 H 2.048 0.005 2 39 6 6 GLU HG2 H 2.266 0.005 2 40 6 6 GLU HG3 H 2.209 0.005 2 41 6 6 GLU CA C 54.180 0.100 1 42 6 6 GLU CB C 25.639 0.100 1 43 6 6 GLU CG C 32.988 0.100 1 44 7 7 SER H H 7.879 0.005 1 45 7 7 SER HA H 4.457 0.005 1 46 7 7 SER HB2 H 4.294 0.005 2 47 7 7 SER HB3 H 3.953 0.005 2 48 7 7 SER CA C 56.367 0.100 1 49 7 7 SER CB C 62.874 0.100 1 50 8 8 ASP H H 8.846 0.005 1 51 8 8 ASP HA H 4.326 0.005 1 52 8 8 ASP HB2 H 2.566 0.005 1 53 8 8 ASP HB3 H 2.566 0.005 1 54 8 8 ASP CA C 54.012 0.100 1 55 8 8 ASP CB C 37.841 0.100 1 56 9 9 GLU H H 8.729 0.005 1 57 9 9 GLU HA H 3.676 0.005 1 58 9 9 GLU HB2 H 1.094 0.005 2 59 9 9 GLU HB3 H 1.617 0.005 2 60 9 9 GLU HG2 H 1.935 0.005 1 61 9 9 GLU HG3 H 1.935 0.005 1 62 9 9 GLU CA C 56.844 0.100 1 63 9 9 GLU CB C 25.776 0.100 1 64 9 9 GLU CG C 33.462 0.100 1 65 10 10 ALA H H 7.727 0.005 1 66 10 10 ALA HA H 3.557 0.005 1 67 10 10 ALA HB H 1.312 0.005 1 68 10 10 ALA CA C 51.718 0.100 1 69 10 10 ALA CB C 16.387 0.100 1 70 11 11 ARG H H 7.641 0.005 1 71 11 11 ARG HA H 3.887 0.005 1 72 11 11 ARG HB2 H 1.798 0.005 1 73 11 11 ARG HB3 H 1.798 0.005 1 74 11 11 ARG HG2 H 1.658 0.005 2 75 11 11 ARG HG3 H 1.528 0.005 2 76 11 11 ARG HD2 H 3.107 0.005 1 77 11 11 ARG HD3 H 3.107 0.005 1 78 11 11 ARG HE H 7.132 0.005 1 79 11 11 ARG CA C 55.778 0.100 1 80 11 11 ARG CB C 27.348 0.100 1 81 11 11 ARG CG C 25.049 0.100 1 82 11 11 ARG CD C 40.677 0.100 1 83 12 12 ARG H H 7.834 0.005 1 84 12 12 ARG HA H 4.043 0.005 1 85 12 12 ARG HB2 H 1.736 0.005 1 86 12 12 ARG HB3 H 1.736 0.005 1 87 12 12 ARG HG2 H 1.652 0.005 2 88 12 12 ARG HG3 H 1.529 0.005 2 89 12 12 ARG HD2 H 3.102 0.005 1 90 12 12 ARG HD3 H 3.102 0.005 1 91 12 12 ARG HE H 7.289 0.005 1 92 12 12 ARG CA C 55.358 0.100 1 93 12 12 ARG CB C 27.548 0.100 1 94 12 12 ARG CG C 24.713 0.100 1 95 12 12 ARG CD C 40.647 0.100 1 96 13 13 CYS H H 7.788 0.005 1 97 13 13 CYS HA H 4.505 0.005 1 98 13 13 CYS HB2 H 3.011 0.005 1 99 13 13 CYS HB3 H 3.011 0.005 1 100 13 13 CYS CA C 54.573 0.100 1 101 13 13 CYS CB C 38.616 0.100 1 102 14 14 TYR H H 8.089 0.005 1 103 14 14 TYR HA H 4.269 0.005 1 104 14 14 TYR HB2 H 2.997 0.005 1 105 14 14 TYR HB3 H 2.997 0.005 1 106 14 14 TYR HD1 H 7.051 0.005 1 107 14 14 TYR HD2 H 7.051 0.005 1 108 14 14 TYR HE1 H 6.772 0.005 1 109 14 14 TYR HE2 H 6.772 0.005 1 110 14 14 TYR CA C 56.928 0.100 1 111 14 14 TYR CB C 35.313 0.100 1 112 15 15 ASN H H 7.822 0.005 1 113 15 15 ASN HA H 4.520 0.005 1 114 15 15 ASN HB2 H 2.888 0.005 2 115 15 15 ASN HB3 H 2.574 0.005 2 116 15 15 ASN HD21 H 7.478 0.005 2 117 15 15 ASN HD22 H 6.819 0.005 2 118 15 15 ASN CA C 50.646 0.100 1 119 15 15 ASN CB C 36.263 0.100 1 120 16 16 ASP H H 7.640 0.005 1 121 16 16 ASP HA H 5.080 0.005 1 122 16 16 ASP HB2 H 3.094 0.005 2 123 16 16 ASP HB3 H 2.565 0.005 2 124 16 16 ASP CA C 48.031 0.100 1 125 16 16 ASP CB C 40.317 0.100 1 126 17 17 PRO HA H 4.400 0.005 1 127 17 17 PRO HB2 H 1.936 0.005 2 128 17 17 PRO HB3 H 2.365 0.005 2 129 17 17 PRO HG2 H 2.000 0.005 1 130 17 17 PRO HD2 H 3.954 0.005 2 131 17 17 PRO HD3 H 3.910 0.005 2 132 17 17 PRO CA C 61.584 0.100 1 133 17 17 PRO CB C 29.626 0.100 1 134 17 17 PRO CG C 24.450 0.100 1 135 17 17 PRO CD C 48.476 0.100 1 136 18 18 LYS H H 8.278 0.005 1 137 18 18 LYS HA H 4.235 0.005 1 138 18 18 LYS HB2 H 1.808 0.005 2 139 18 18 LYS HB3 H 1.852 0.005 2 140 18 18 LYS HG2 H 1.418 0.005 2 141 18 18 LYS HG3 H 1.363 0.005 2 142 18 18 LYS HD2 H 1.662 0.005 1 143 18 18 LYS HD3 H 1.662 0.005 1 144 18 18 LYS HE2 H 2.967 0.005 1 145 18 18 LYS HE3 H 2.967 0.005 1 146 18 18 LYS CA C 53.310 0.100 1 147 18 18 LYS CB C 29.145 0.100 1 148 18 18 LYS CG C 22.326 0.100 1 149 18 18 LYS CD C 26.011 0.100 1 150 18 18 LYS CE C 39.507 0.100 1 151 19 19 CYS H H 7.968 0.005 1 152 19 19 CYS HA H 4.500 0.005 1 153 19 19 CYS HB2 H 3.570 0.005 2 154 19 19 CYS HB3 H 3.535 0.005 2 155 19 19 CYS CA C 54.152 0.100 1 156 19 19 CYS CB C 41.021 0.100 1 157 20 20 SER H H 8.649 0.005 1 158 20 20 SER HA H 4.478 0.005 1 159 20 20 SER HB2 H 3.969 0.005 2 160 20 20 SER HB3 H 3.822 0.005 2 161 20 20 SER CA C 55.358 0.100 1 162 20 20 SER CB C 61.163 0.100 1 163 21 21 ASP H H 7.969 0.005 1 164 21 21 ASP HA H 4.622 0.005 1 165 21 21 ASP HB2 H 2.700 0.005 1 166 21 21 ASP HB3 H 2.700 0.005 1 167 21 21 ASP CB C 38.777 0.100 1 168 22 22 SER H H 8.122 0.005 1 169 22 22 SER HA H 4.482 0.005 1 170 22 22 SER HB2 H 3.761 0.005 2 171 22 22 SER HB3 H 3.737 0.005 2 172 22 22 SER CA C 55.498 0.100 1 173 22 22 SER CB C 61.780 0.100 1 174 23 23 VAL H H 7.746 0.005 1 175 23 23 VAL HA H 4.207 0.005 1 176 23 23 VAL HB H 1.897 0.005 1 177 23 23 VAL HG1 H 0.962 0.005 2 178 23 23 VAL HG2 H 0.917 0.005 2 179 23 23 VAL CA C 58.948 0.100 1 180 23 23 VAL CG1 C 18.798 0.100 1 181 23 23 VAL CG2 C 18.554 0.100 1 182 24 24 CYS H H 8.640 0.005 1 183 24 24 CYS HA H 4.511 0.005 1 184 24 24 CYS HB2 H 3.229 0.005 2 185 24 24 CYS HB3 H 2.704 0.005 2 186 24 24 CYS CA C 52.441 0.100 1 187 24 24 CYS CB C 38.017 0.100 1 188 25 25 LYS H H 8.098 0.005 1 189 25 25 LYS HA H 4.141 0.005 1 190 25 25 LYS HB2 H 1.801 0.005 2 191 25 25 LYS HB3 H 1.672 0.005 2 192 25 25 LYS HG2 H 1.386 0.005 1 193 25 25 LYS HG3 H 1.386 0.005 1 194 25 25 LYS HD2 H 1.615 0.005 1 195 25 25 LYS HD3 H 1.615 0.005 1 196 25 25 LYS HE2 H 2.966 0.005 1 197 25 25 LYS HE3 H 2.966 0.005 1 198 25 25 LYS CA C 55.077 0.100 1 199 25 25 LYS CB C 31.194 0.100 1 200 25 25 LYS CG C 22.109 0.100 1 201 25 25 LYS CD C 26.410 0.100 1 202 25 25 LYS CE C 39.230 0.100 1 stop_ save_