JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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生物化学

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Published: January 10, 2018
doi:
10.3791/56953
, ,
1Signal Transduction Laboratory, National Institutes of Environmental Health Sciences, Department of Health and Human Services,National Institutes of Health

Summary

该方法描述了用 x 射线晶体学和小角 x 射线散射 (SAXS) 进行结构测定的重组 Nsa1 的克隆、表达和纯化, 适用于其他蛋白质的混合结构分析包含有序和无序的域。

Abstract

从酿酒酵母 (酵母)中 Nsa1 核糖体组装因子的全长结构的测定是有挑战性的, 因为蛋白质紊乱和蛋白酶不稳定的 c-末端。本文介绍了从酿酒酵母中纯化重组 Nsa1 的方法, 通过 x 射线晶体学和 SAXS 进行结构分析。利用 X 射线晶体学的方法求解了 Nsa1 的有序 N 端 WD40 域的结构, 然后利用 SAXS 解决了 Nsa1 的 c-末端结构。溶液散射数据是从全长 Nsa1 中收集的。从 WD40 域的高分辨率晶体结构计算了理论散射振幅, 然后结合刚体和从头算模型揭示了 Nsa1 的 c-终点. 通过这种混合方法, 重建了整个蛋白质的第四纪结构。这里提出的方法应该是一般适用于混合结构确定的其他蛋白质组成的混合结构和非结构化领域。

Introduction

核糖体是大型的核糖机器, 它能在所有活细胞中实现将 mRNA 转化为蛋白质的重要作用。核糖体是由两个亚基组成的一个复杂的过程中, 称为核糖体生物1,2,3,4。真核核糖体组装依赖于数百个必要的核糖体组装因子2,3,5。Nsa1 (Nop7 相关的 1) 是一个真核核糖体组装因子, 是专门为生产大核糖体亚基6, 并称为 WD 重复包含 74 (WDR74) 在较高的生物体7。WDR74 已被证明是需要的囊胚形成在小鼠8和 WDR74 启动子经常突变的癌细胞9。然而, Nsa1/WDR74 在核糖体组装中的作用和精确机制仍然是未知数。为了开始揭示 Nsa1/WDR74 在真核核糖体成熟过程中的作用, 进行了多种结构分析, 包括 x 射线晶体学和小角 x 射线散射 (SAXS)10。

x-射线结晶学、核磁共振 (NMR) 光谱学、电子显微镜和 SAXS 都是研究大分子结构的重要技术。大分子的大小、形状、可用性和稳定性影响到一种特定的大分子最适合的结构生物学方法, 然而通过 so-called “混合” 方法将多种技术结合起来, 正成为日益受益的工具11。特别是 x 射线结晶学和 SAXS 是强有力和补充的方法为大分子的结构决心12。

晶体学提供了高分辨率的原子结构, 从小分子到大型蜂窝机械, 如核糖体, 并在理解蛋白质和其他生物功能方面取得了许多突破。大分子13。此外, 结构药物设计利用计算方法为分子对接提供了晶体结构的能量, 为药物发现和开发14增添了一个关键的维度。尽管它具有广泛的适用性, 但由于晶体包装可以被阻碍或电子密度图可能不完整或质量较差, 因此, 由晶体学来评估的灵活性和无序系统具有挑战性。相反, SAXS 是一种解决和低分辨率结构方法, 能够描述从无序循环和总站到固有无序的蛋白质的灵活系统12,15,16。考虑到它与范围广泛的粒子大小12兼容, SAXS 可以与晶体学协同工作, 以扩大可以通过结构研究解决的生物问题的范围。

Nsa1 是适合混合结构的方法, 因为它包含一个合理的 WD40 域, 其次是一个功能, 但灵活的 C 总站, 这是不服从 X 射线晶体学方法。以下是S. 酿酒酵母Nsa1 的克隆、表达和纯化的协议, 用于 X 射线晶体学和 SAXS 的混合结构测定。本协议可用于研究其他蛋白质的结构, 它们由有序和无序区域的组合组成。

Protocol

1. 重组蛋白的生产和纯化1 Nsa1 表达质粒的设计与克隆 获取或购买S. 酿酒酵母基因组 DNA。 PCR 放大的目标序列 Nsa1 (Nsa1FL, 残留 1-463) 和 c-终端截断 Nsa1 (Nsa1ΔC, 残留物 1-434) 与适当的底漆使用基因组 DNA 分离从S. 酿酒酵母和熔化温度大约60° c 以延长时间 1-2 min。以下引物用于放大 Nsa1:SC_Nsa1_FLFw:CGCCAAAGGCCTATGAGGTTACTAGTCAGCTGTGTGGATAGSC_Nsa1_FLRv:AATGCAGCGGCCG…

Representative Results

Nsa1 是 PCR 扩增从酿酒酵母基因组 DNA 和亚成一个载体, 其中含有 n-端 6 x-氨酸的亲和标记, 其次是碱性蛋白和 TEV 蛋白酶的网站。Nsa1 被转化成了大肠杆菌BL21 (DE3) 细胞, 在诱导与 IPTG 和生长25° c (图 1A) 后获得了高的蛋白质表达率。Nsa1 在固定化钴亲和树脂上进行亲和纯化 , 其次是 TEV 蛋白酶的碱性蛋白 , 最后通过大小排斥色谱 (<strong class="x…

Discussion

使用这个协议, 重组 Nsa1 从S. 酿酒酵母是由 X 射线晶体学和 SAXS 的结构研究产生的。Nsa1 在溶液中表现良好, 并以多种晶体形式结晶。在这些晶体的优化过程中, 发现 Nsa1 的 C 端对蛋白酶的降解非常敏感。高分辨率, 正交晶体的形式, 只能与 c 终端的 Nsa1 的截断变种, 可能是因为灵活的 c 总站 Nsa1 防止晶体包装。Nsa1 的结构由 x-射线结晶学解决以高分辨率, 但 C 末端不可能被修造在二者之一水晶?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

在先进的光子源 (APS), 阿贡国家实验室, 在东南区域协作访问组 (SER CAT) 22 ID 和 22 BM 光束收集了衍射数据。SAXS 数据是收集在女巫光束在先进的光源 (ALS), 劳伦斯伯克利国家实验室。我们要感谢女巫光束的工作人员对远程数据收集和处理的帮助。我们感谢国家环境健康科学研究所 (NIEHS) 质谱研究和支持小组帮助确定蛋白质领域的边界。这项工作得到了美国国立卫生研究院的校内研究项目的支持;美国国家环境健康科学研究所 (NIEHS) (齐亚 ES103247 至 r.e.s.) 和加拿大卫生研究所 (研究院, 146626 至. P)。APS 的使用得到了美国能源部, 科学办公室, 基础能源科学办公室的支持, 合同 No。W-31-109-Eng-38使用先进光源 (ALS) 的支持下, 美国能源部科学办公室, 基础能源科学办公室, 根据合同 No。DE-AC02-05CH11231对女巫 SAXS 光束的额外支持来自国家卫生研究院项目米诺斯 (R01GM105404) 和高端仪器补助金 S10OD018483。我们还要感谢安德里亚. Dr. 和萨拉. 安德斯对这份手稿的批判性阅读。

Materials

Molecular Cloning of Nsa1
pMBP2 parallel vector Sheffield et al, Protein Expression and Purification 15, 34-39 (1999) We used a modified version of pMBP2 which included an N-terminal His-tag (pHMBP)
S. cerevisiae genomic DNA ATCC 204508D-5
Primers for cloning Nsa1
SC_Nsa1_FLFw IDT CGC CAA AGG CCT
ATGAGGTTACTAGTCAGCTGTGT
GGATAG
SC_Nsa1_FLRv IDT AATGCAGCGGCCGCTCAAATTTT
GCTTTTCTTACTGGCTTTAGAAGC
AGC
SC_Nsa1_DeltaCFw IDT GGGCGCCATGGGATCCATGAGG
TTACTAGTCAGCTGTGTGG
SC_Nsa1_DeltaCRv IDT GATTCGAAAGCGGCCGCTTAAAC
CTTCCTTTTTTGCTTCCC
Recombinant Protein Production and Purification of Nsa1
Escherichia coli BL21 (DE3) Star Cells Invitrogen C601003
pMBP- NSA1 and various truncations Lo et al., 2017
Selenomethionine Molecular Dimensions MD12-503B
IPTG, Dioxane-Free Promega V3953
EDTA Free Protease Inhibitor Cocktail Sigma-Aldrich 4693159001
Sodium Chloride Caledon Laboratory Chemicals 7560-1-80
Magnesium Chloride hexahydrate Sigma-Aldrich M2670
Tris Buffer, 1 M pH7.5 KD Medical RGF-3340
Glycerol Invitrogen 15514-029
beta-mercaptoethanol Sigma M6250
1M Imidazole, pH 8.0 Teknova I6980-06
Talon Affinity Resin Clonetech 635503
Amicon Ultra 15 mL Centrifugal Filter (MWCO 10K) Millipore UFC901024
HiLoad 16/600 Superdex 200 Prep Grade Gel Filtration Column GE-Healthcare 28989335
TEV Protease Prepared by NIEHS Protein Expression Core Expression plasmid provided by NCI (Tropea et al. Methods Mol Biology, 2009)
4-15% Mini-PROTEAN TGX Precast Protein Gels BioRad 456-8056
Crystallization, Proteolytic Screening
Crystal Screen Hampton Research HR2-110
Crystal Screen 2 Hampton Research HR2-112
Salt Rx Hampton Research HR2-136
Index  Screen Hampton Research HR2-144
PEG/Ion Screen Hampton Research HR2-139
JCSG+ Molecular Dimensions MD1-37
Wizard Precipitant Synergy  Molecular Dimensions MD15-PS-T
Swissci 96-well 3-drop UVP sitting drop plates TTP Labtech 4150-05823
3inch Wide Crystal Clear Sealing Tape Hampton Research HR4-506
Proti-Ace Kit Hampton Research HR2-429
PEG 1500 Molecular Dimensions MD2-100-6
PEG 400 Molecular Dimensions MD2-100-3
HEPES/sodium hydroxide pH 7.5 Molecular Dimensions MD2-011-
Sodium Citrate tribasic Molecular Dimensions MD2-100-127
22 mm x 0.22 mm Siliconized Coverslides Hampton Research HR3-231
24 Well Plates with sealant (VDX Plate with Sealant) Hampton Research HR3-172
 18 mM Mounted Nylon Loops (0.05 mm to 0.5 mM) Hampton Research HR4-945, HR4-947, HR4-970, HR4-971
Seed Bead Kit Hampton Research HR2-320
Magnetic Crystal Caps Hampton Research HR4-779
Magnetic Cryo Wand Hampton Research HR4-729
Cryogenic Foam Dewar Hampton Research HR4-673
Crystal Puck System MiTeGen M-CP-111-021
Full Skirt 96 well Clear Plate VWR 10011-228
AxyMat Sealing Mat VWR 10011-130
Equipment
UVEX-m JAN Scientific, Inc.
Nanodrop Lite Spectrophotometer Thermo-Fisher
Mosquito Robot TTP Labtech
Software/Websites
HKL2000 Otwinoski and Minor, 1997
Phenix Adams et al., 2010
Coot Emsley et al., 2010
ATSAS Petoukhov et al., 2012 https://www.embl-hamburg.de/biosaxs/atsas-online/
Scatter Rambo and Tainer, 2013
Pymol The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.
BUNCH Petoukhov and Svergun, 2005
CRYSOL Svergun et al, 1995
PRIMUS Konarev et al, 2003
EOM Tria et al, 2015
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Tags

X-ray CrystallographySmall Angle X-ray Scattering (SAXS)Structural AnalysisProtein StructureRecombinant ProteinUnstructured RegionsNsa1S. CerevisiaeHybrid Structural AnalysisProtein FunctionProtein DynamicsProtein AggregationPurificationAffinity ChromatographyGel FiltrationSDS-PAGETEV ProteaseCentrifugal Filtration

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Lo, Y., Pillon, M. C., Stanley, R. E. Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae. J. Vis. Exp. (131), e56953, doi:10.3791/56953 (2018).
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