根拠: assay for oligomerization, Peptide concentration was assessed over a fifty-fold range of concentrations in 1D 1H NMR spectra from 0.1 mM up to 5.0 mM. While all concentrations below and ...根拠: assay for oligomerization, Peptide concentration was assessed over a fifty-fold range of concentrations in 1D 1H NMR spectra from 0.1 mM up to 5.0 mM. While all concentrations below and inclusive of 1.0 mM peptide were acceptable - free of dispersion and broadening effects across multiple weeks in solution - 0.5 mM was selected as the optimal choice for further analysis with optimal resolution.
タイプ
名称
対称操作
数
identity operation
1_555
1
NMR アンサンブル
データ
基準
コンフォーマー数 (登録 / 計算)
25 / 1000
structures with the lowest energy
代表モデル
モデル #1
lowest energy
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要素
#1: タンパク質・ペプチド
Galanin
分子量: 3159.454 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) Homo sapiens (ヒト) / 参照: UniProt: P22466
研究の焦点であるリガンドがあるか
N
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実験情報
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実験
実験
手法: 溶液NMR
NMR実験
Conditions-ID
Experiment-ID
Solution-ID
Sample state
Spectrometer-ID
タイプ
1
1
1
isotropic
1
1H1DNMRSpectrum
1
2
1
isotropic
1
2D 1H-1H COSY
1
3
1
isotropic
1
2D 1H-1H TOCSY
1
4
1
isotropic
1
2D 1H-1H ROESY
NMR実験の詳細
Text: The authors state the following regarding the geometric deviations with this ensemble: With most of the hydrogen bonds in the N-terminus of the peptide bifurcated, or even more, they are ...Text: The authors state the following regarding the geometric deviations with this ensemble: With most of the hydrogen bonds in the N-terminus of the peptide bifurcated, or even more, they are confident that the ensemble represents the time-averaged structure of a peptide that is rapidly interconverting turns of alpha, 310-, and pi-helix. Their shift assignments were largely unambiguous and can be found in BMRB entry 31032. The "irregular helix restraints" used (and described in the manuscript and its references) were employed to allow the ensemble to sample multiple arrangements during simulated annealing. By definition, this ensemble existing as an average will display a significant degree of geometric irregularity, particularly with regard the backbone and sidechain outliers as compared to statistical norms. Further, the clashscore level is quite high. This appears to be a consequence of the very high density of ROE-derived distance restraints in the N-terminus of the peptide, which were maximally relaxed to account for any uncertainty in their measurement throughout several rounds of validation and re-calculation. Tight sidechain packing and compression seems to be the dictated outcome of our data. Much of their study is concerned with compact hydrophobic packing in this sequence - this full-length peptide is indeed much more rigid across the stretch of residues it shares with fragment peptides described in entries 7S3O, 7S3Q, and 7S3R. Together with the compactness of the region of the sequence, the clashes appear to be a consequence of several averaged, coiling, helical conformers all existing on the NMR timescale. This is discussed at further length in the citation affiliated with this deposition
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試料調製
詳細
タイプ: lyophilized powder 内容: 0.5 mM hGal(1-30)NH2, 4.14 mM [U-2H] sodium acetate, 5.86 mM [U-2H] acetic acid, 95% H2O/5% D2O 詳細: peptide was suspended and dissolved into deuterated acetate buffer, which had been prepared in 95:5 H2O:D2O to within 0.02 units of pH 4.6 Label: 1 / 溶媒系: 95% H2O/5% D2O
試料
濃度 (mg/ml)
構成要素
Isotopic labeling
Solution-ID
0.5mM
hGal(1-30)NH2
naturalabundance
1
4.14mM
sodiumacetate
[U-2H]
1
5.86mM
aceticacid
[U-2H]
1
試料状態
イオン強度: sample was dissolved in 10 mM sodium acetate-d3/acetic acid-d4 buffer at pH 4.6; no additional additives were present. mM Label: 1 / pH: 4.6 / PH err: 0.02 / 圧: 1 atm / 温度: 298 K
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NMR測定
NMRスペクトロメーター
タイプ: Bruker AVANCE III / 製造業者: Bruker / モデル: AVANCE III / 磁場強度: 600 MHz / 詳細: TCI H-C/N-D Cryoprobe
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解析
NMR software
名称
開発者
分類
CNS
BrungerA. T. et.al.
精密化
CNS
Brunger, Adams, Clore, Gros, NilgesandRead
structurecalculation
MestreLab (Mnova / MestReNova / MestReC)
MestreLab
chemicalshiftassignment
MestreLab (Mnova / MestReNova / MestReC)
MestreLab
peakpicking
精密化
手法: simulated annealing / ソフトェア番号: 1
代表構造
選択基準: lowest energy
NMRアンサンブル
コンフォーマー選択の基準: structures with the lowest energy 計算したコンフォーマーの数: 1000 / 登録したコンフォーマーの数: 25