SASDEN2: Labeled Nuclear Localization Signal from the inner nuclear membra...

基本情報

登録情報

データベース: SASBDB / ID: SASDEN2

試料

Labeled Nuclear Localization Signal from the inner nuclear membrane protein HEH2 (NLS-Alexa488/Alexa594) without denaturant

• Inner nuclear membrane protein HEH2 (protein), NLS-488/594, Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
• Alexa Fluor™ 594 C5 Maleimide (other), Alexa594
• Alexa Fluor™ 488 C5 Hydroxylamine (other), Alexa488

機能・相同性

分子機能:

nuclear membrane organization / nuclear inner membrane / nuclear periphery / nuclear envelope / chromatin binding

ドメイン・相同性:

Heh2/Src1-like / HeH/LEM domain / HeH/LEM domain / Man1/Src1, C-terminal / MAN1, winged-helix domain / Man1-Src1p-C-terminal domain / LEM/LEM-like domain superfamily

構成要素:

Inner nuclear membrane protein HEH2

• 生物種: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (パン酵母)
• 引用: ジャーナル: Proc Natl Acad Sci U S A / 年: 2017; タイトル: Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.; 著者: Gustavo Fuertes / Niccolò Banterle / Kiersten M Ruff / Aritra Chowdhury / Davide Mercadante / Christine Koehler / Michael Kachala / Gemma Estrada Girona / Sigrid Milles / Ankur Mishra / Patrick R Onck / Frauke Gräter / Santiago Esteban-Martín / Rohit V Pappu / Dmitri I Svergun / Edward A Lemke / ; 要旨: Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values and For chemically denatured proteins we obtain mutual consistency in our inferences based on and , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between and that is amplified in the absence of denaturants. Therefore, joint assessments of and combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

登録者

• Gustavo Fuertes Vives (Institute of Biotechnology CAS v.v.i.)

モデル

試料

• 試料: 名称: Labeled Nuclear Localization Signal from the inner nuclear membrane protein HEH2 (NLS-Alexa488/Alexa594) without denaturant; 試料濃度: 1.50-7.50 / Entity id: 1251 / 1252 / 1253
• バッファ: 名称: 25 mM HEPES, 150 mM NaCl, 10 mM DTT / pH: 7.4

要素 #1251

名称: NLS-488/594 / タイプ: protein / 記述: Inner nuclear membrane protein HEH2 / 分子量: 5.406 / 分子数: 1
由来: Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
参照: UniProt: Q03281
配列:

ACETNKRKRE QISTDNEAKM QIQEEKSPKK KRKKRSSKAN KPPEUA

• 要素 #1252: 名称: Alexa594 / タイプ: other / 記述: Alexa Fluor™ 594 C5 Maleimide / 分子量: 0.886 / 分子数: 1
• 要素 #1253: 名称: Alexa488 / タイプ: other / 記述: Alexa Fluor™ 488 C5 Hydroxylamine / 分子量: 0.7 / 分子数: 1

実験情報

• ビーム: 設備名称: PETRA III EMBL P12 / 地域: Hamburg / 国: Germany / 線源: X-ray synchrotron / 波長: 0.1 Å / スペクトロメータ・検出器間距離: 3 mm
• 検出器: 名称: Pilatus 2M

スキャン

タイトル: Labeled Nuclear Localization Signal from the inner nuclear membrane protein HEH2 (NLS-Alexa488/Alexa594) without denaturant
測定日: 2014年1月25日 / セル温度: 23 °C / 照射時間: 0.05 sec. / フレーム数: 20 / 単位: 1/nm /

	Min	Max
Q	0.0199	4.5094

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 1309 /

	Min	Max
Q	0.0884369	3.53456
P(R) point	1	1309
R	0	7.29

結果

カーブのタイプ: merged
コメント: The protein contains a penultimate non-canonical amino acid p-acetylphenylalanine (207 Da) that is represented as U (selenocysteine, 168 Da) in the amino acid sequence for the entry. Therefore, the calculated MW from sequence (MW(expected)) must be adjusted accordingly (ca. 40 Da).

	実験値	Porod
分子量	8.5 kDa	-
体積	-	9.9 nm3

	P(R)	P(R) error	Guinier	Guinier error
前方散乱 I0	224.4	3.5	223.9	2.9
慣性半径, Rg	2.166 nm	0.03	2 nm	0.2

	Min	Max
D	-	7.29
Guinier point	27	236