概要

重组卡2 +- 研究色氨酸 - ANS FRET 的色氨酸残留物的化学修饰 - ATPase N 域

Published: October 09, 2021
doi:

概要

ANS 与 Ca2+– ATPase 重组 N 域绑定。荧光光谱在激发时以 295 nm 的波长显示类似 FRET 的图案。NBS 调解的 Trp 化学修饰抑制了 N 域的荧光,这导致 Trp 残留物和 ANS 之间没有能量转移 (FRET)。

Abstract

沙科/内质 Ca2+-ATPase (SERCA) 是一种 P 型 ATPase,在各种构象中结晶。尽管如此,仍可从孤立的重组域获取详细的功能信息。工程 (Trp552Leu 和 Tyr587Trp) 重组核苷酸绑定域 (N 域) 显示配体绑定后荧光淬火。一种外在的氟磷,即8-阿尼利诺-1-纳布他烯硫酸盐(ANS),通过静电和疏水性与阿格、赫斯、阿拉、卢和菲残留物的相互作用,与核苷酸结合。当兴奋的波长(+)为370纳米时,荧光强度的增加就证明了ANS的结合。然而,当兴奋在 \295 nm 时,荧光强度的增加似乎与 N 域内在荧光的淬火相伴而生。荧光光谱显示一个类似Föster共振能量转移(FRET)的模式,从而暗示了Trp-ANS FRET对的存在,这似乎由Tyr587Trp和ANS之间的短距离(+20+)支撑。本研究描述了由 N-溴素酰胺 (NBS)调解的 Trp-ANS FRET 对的 Trp 化学修饰(和荧光淬火)分析。在化学改性 N 域中,ANS 荧光在 295 nm 的兴奋时增加,类似于在 370 nm 时兴奋时增加。因此,NBS 调解的 Trp 残留物的化学修饰可用于探索 Trp 和 ANS 之间缺乏 FRET。在没有TRP荧光的情况下,不应观察ANS荧光的增加。NBS 对蛋白质中的 Trp 残留物进行化学修饰可能有助于检查接近绑定 ANS 的 Trp 残留物之间的 FRET。这种检测在使用其他氟化物时可能也很有用。

Introduction

Füster共振能量转移(FRET)已成为确定分子结构之间的距离后,结合或相互作用的蛋白质结构和功能研究1,2,3,4的标准技术。在P型ATPases中,FRET被用来研究SARCO内质视网膜Ca 2+-ATPase(SERCA)2、5、6、7、8的结构波动,例如,FRET7对整个蛋白质的结构波动进行了分析。

FRET捐赠者是多种多样的,从小荧光(外在)分子到荧光蛋白9,10。色氨酸(Trp)残留物(由于其荧光)有助于识别蛋白质氨基酸序列11,12的结构变化。Trp的荧光强度在很大程度上取决于其周围环境性。利甘结合通常产生蛋白质/酶15,16的结构重新排列。如果Trp位于或位于蛋白质结合部位附近,结构波动通常会影响Trp暴露在13、14等水平:因此,极性的变化导致Trp荧光强度13,14的淬火。因此,Trp 的荧光特性对于对酶进行配体结合研究很有用。其他物理现象也可能导致Trp荧光淬火17,18,19,20,例如,FRET和中等极性的变化。能量从兴奋状态的Trp转移到氟也具有潜在的应用,例如,在蛋白质21中小配体的亲和力测定。事实上,Trp在FRET研究中主要用作22、23、24等蛋白质的荧光捐献者,例如,在terbium(Tb3+)FRET研究中,Trp残留物经常被用作能量转移到Tb 3+25、26、27的天线。Trp由于其在蛋白质结构中固有的构成特性,与其他FRET捐赠者相比具有各种优势,从而消除了可能影响所研究蛋白质24的功能/结构的预制过程的需要。因此,在蛋白质结构研究13、14、19、28中,对辐射衰变(由蛋白质结构重组引起的能量转移和中等极性变化)的识别对于得出关于配体结合的准确结论非常重要。

在蛋白质结构研究中,一种外在的氟磷,即8-阿尼利诺-1-纳布他烯硫酸盐(ANS),主要用于与蛋白质折叠/展开有关的实验。ANS与原生状态的蛋白质/酶结合,通常在基板31、32、33的结合部位:ANS荧光量子产量(+F)的增加(即荧光强度的增加)是由刺激蛋白质在\370纳米时,ANS与Arg的适当相互作用和他的残留物在疏水口袋发生34,35,36,37。在各种研究中, 已报告TRP残留物(捐赠者)和ANS(接受者)之间的FRAT(当在280-295纳米内激动人心时)的发生,其依据如下:1) Trp 的荧光发射光谱和 ANS 的激发光谱重叠,2) 确定一个或多个 Trp 残留物与 ANS 之间的适当距离用于能量转移, 3) 高ANS量子产量时,结合在蛋白质口袋,和4)特征FRET模式在蛋白质的荧光光谱中存在ANS 3,17,27,37,38。

最近,使用工程重组N域40、41、42、43、44、45、46等对SERCA和其他P型ATPass的核苷酸结合域(N域)进行了配体结合。SERCA N域的分子工程已用于将唯一的Trp残留物(Trp552Leu)移动到一个更动态的结构(Tyr587Trp),该结构靠近核苷酸结合部位,其中荧光变异(淬火)可用于监测配体结合34时的结构变化。实验结果表明,ANS与纯重组剂SERCA N-domain34中的核苷酸结合部位(作为ATP)结合。有趣的是,ANS 荧光在激发时以 295 nm 的激发度与 N 域结合时增加,而 N 域的内在荧光减少34,从而产生一个 FRET 模式,表明 Trp-ANS FRET 对的形成。

已建议使用 NBS 通过对改性蛋白质的吸收分析来确定蛋白质 47中的 Trp 残留物含量。NBS将Trp高度吸收的内陆组修改为吸收性较小的奥辛多莱47,48。这导致Trp荧光财产损失(淬火)40。因此,NBS 调解的Trp残留物的化学修饰可以用作在FRET假设时定义Trp(作为供体)的作用的测定。

本协议将国家统计局在 SERCA 工程重组 N 域中唯一 Trp 残留物的化学修改描述为蛋白质模型。实验结果表明,在化学NBS改性N-domain34中,ANS荧光强度仍在增加,缺乏内在荧光。因此,当与 N 域34、40、49绑定时该检测有助于证明 Trp 残留物和 ANS 之间没有 FRET。因此,这种检测(Trp的NBS化学修饰)有助于证明Trp-ANS FRET对在蛋白质中的存在。

Protocol

1. 确定(在西里科)的ANS和SERCAN域互动 使用首选的蛋白质建模软件50进行分子建模,生成蛋白质(SERCA N-Domain)的三维(3D)结构。 使用首选分子结构软件51识别形成核苷酸结合位点的氨基酸残留物,确定Arg和Lys残留物的存在:这些是ANS结合和增加荧光强度(量子产量)所必需的。 执行分子对接(使用首选对接软?…

Representative Results

分子对接显示ANS通过静电和疏水相互作用与N域核苷酸结合部位的结合(图1)。Trp 残留物和 ANS(与核苷酸结合部位)之间的分子距离 (20+) 支持 FRET 的发生(图1)。设计(工程)重组N域通过亲和色谱仪(图2)获得高纯度,适合荧光实验。ANS-N 域复合物的荧光光谱在激发时显示类似 FRET 的图案,频率为 ±295 nm(?…

Discussion

ANS-N 域复合物的荧光光谱在 295 nm 的兴奋时显示类似 FRET 的模式,而 Trp 残留物和 ANS 之间的分子距离 (20 +) 似乎支持 FRET 的发生(图1)。NBS 的 Trp 化学修饰导致荧光 N 域变小(图 3B,频谱 f):因此,能量转移是不可能的。ANS 荧光光谱与未修改的 N 域相似,当兴奋在 295 nm (图 3A 和 C)时。

因…

開示

The authors have nothing to disclose.

Acknowledgements

这项工作部分由FAI-UASLP赠款编号C19-FAI-05-89.89和CONACYT赠款编号316463(阿波约斯前 拉:福塔莱西门托和曼特尼门托基础设施研究公司,乌索科门和卡帕西塔奇-恩特克尼卡2021年)。作者感谢朱利安·马塔-莫拉莱斯在视频版中的技术帮助。

Materials

Acrylamide Bio-Rad 1610107 SDS-PAGE
Ammonium persulfate Bio-Rad 1610700 SDS-PAGE
8-Anilino-1-naphthalenesulfonic acid Sigma-Aldrich A1028 Fluorophore
Bis-acrylamide Bio-Rad 1610125 SDS-PAGE
N-Bromosuccinimide Sigma-Aldrich B81255 Chemical modification
N,N-dimethylformamide J.T. Baker 9213-12 Stock solution preparation
Fluorescein isothiocyanate Sigma-Aldrich F7250 Chemical fluorescence label
Fluorescence cuvette Hellma Z801291 Fluorescence assay
Fluorescence Spectrofluorometer Shimadzu RF 5301PC Fluorescence assay
HisTrap™ FF GE Healtcare 11-0004-59 Protein purification
IPTG, Dioxane free American Bionalytical AB00841-00010 Protein expression
Imidazole Sigma-Aldrich I5513-25G Protein purification
LB media Fisher Scientific 10000713 Cell culture
Pipetman L P10L Gilson FA10002M Fluorescence assay
Pipetman L P100L Gilson FA10004M Fluorescence assay
Pipetman L P200L Gilson FA10005M Fluorescence assay
Pipetman L P1000L Gilson FA10006M Fluorescence assay
Pipetman L P5000L Gilson FA10007 Fluorescence assay
Precision plus std Bio-Rad 1610374 SDS-PAGE
Sodium dodecyl sulphate Bio-Rad 1610302 SDS-PAGE
Sodium phosphate dibasic J.T. Baker 3828-19 Buffer preparation
Sodium phosphate monobasic J.T. Baker 3818-01 Buffer preparation
Syringe filter 0.2 um Millipore GVWP04700 Solution filtration
Temed Bio-Rad 1610801 SDS-PAGE
Tris Bio-Rad 1610719 SDS-PAGE

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記事を引用
Sampedro, J. G., Cataño, Y. Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET. J. Vis. Exp. (176), e62770, doi:10.3791/62770 (2021).

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