分子量: 2941.242 Da / 分子数: 1 / 断片: Extracellular Topological domain / 由来タイプ: 合成 詳細: 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 ...詳細: 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. 由来: (合成) Homo sapiens (ヒト) / 参照: UniProt: P08588
配列の詳細
AUTHORS STATE THAT THIS IS AN ANALOG OF THE FRAGMENT 197-221 OF BETA-1 ADRENORECEPTOR.
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実験情報
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実験
実験
手法: 溶液NMR / 詳細: Analog of the fragment 197-221 of 1- adrenoreceptor
NMR実験
Conditions-ID
Experiment-ID
Solution-ID
タイプ
1
1
2
2D 1H-13C HSQC aliphatic
1
2
2
2D 1H-13C HSQC aromatic
1
3
2
2D DQF-COSY
1
4
2
2D 1H-1H NOESY
1
5
2
2D 1H-1H TOCSY
1
6
1
2D 1H-1H NOESY
1
7
1
2D 1H-1H TOCSY
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試料調製
詳細
Solution-ID
内容
溶媒系
1
2 mM protein, 1 mM sodium azide, 20 mM [U-100% 2H] sodium acetate, 90% H2O/10% D2O
90% H2O/10% D2O
2
2 mM protein, 1 mM sodium azide, 20 mM [U-100% 2H] sodium acetate, 100% D2O
100% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
2mM
entity-1
1
1mM
sodium azide-2
1
20mM
sodium acetate-3
[U-100% 2H]
1
2mM
entity-4
2
1mM
sodium azide-5
2
20mM
sodium acetate-6
[U-100% 2H]
2
試料状態
イオン強度: 22 / pH: 5.5 / 圧: ambient / 温度: 303 K
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NMR測定
NMRスペクトロメーター
タイプ: Bruker DPX / 製造業者: Bruker / モデル: DPX / 磁場強度: 500 MHz
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解析
NMR software
名称
バージョン
開発者
分類
XwinNMR
1.3
BrukerBiospin
collection
XwinNMR
1.3
BrukerBiospin
解析
CARA
1.9.5
KellerandWuthrich
chemicalshiftassignment
CARA
1.9.5
KellerandWuthrich
peakpicking
CYANA
3
Guntert, MumenthalerandWuthrich
構造決定
CYANA
Guntert, MumenthalerandWuthrich
精密化
精密化
手法: torsion angle dynamics / ソフトェア番号: 1
NMR constraints
NOE constraints total: 182 / NOE intraresidue total count: 70 / NOE long range total count: 24 / NOE medium range total count: 28 / NOE sequential total count: 60
代表構造
選択基準: fewest violations
NMRアンサンブル
Average torsion angle constraint violation: 1.3 ° / コンフォーマー選択の基準: target function / 計算したコンフォーマーの数: 100 / 登録したコンフォーマーの数: 20 / Maximum lower distance constraint violation: 0.01 Å / Maximum torsion angle constraint violation: 8 ° / Maximum upper distance constraint violation: 0.3 Å