概要

结合干湿实验室技术指导大型卷曲螺旋含有蛋白质的结晶

Published: January 06, 2017
doi:

概要

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Abstract

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

Introduction

通过X射线晶体结构测定作出了现代生物学的各个领域基础性的贡献;提供支持生活以及他们如何与在各种情况下彼此相互作用的大分子的原子视图;让我们来了解导致疾病和提供机会来合理设计药物来治疗疾病的机制。晶体早已确定大分子结构的主要实验技术,目前占结构数据库(www.rcsb.org)的89.3%。这种技术有很多优点,包括非常高的分辨率的潜力,以可视化的大分子具有宽范围的尺寸,相对容易收集数据,并以可视化的大分子如何与溶剂以及配体相互作用的机会的能力。

尽管在重组蛋白表达1,2,PUR众多技术改进ification 3,并用于产生这些系统4,在结晶过程中的最大障碍分子生物学仍然生长衍射质量晶体的能力这一直是尤其如此,它含有大量的卷曲螺旋结构域的蛋白。据估计,所有氨基酸的多达5%的卷曲线圈5,6内发现的,使之成为一个非常常见的结构特征7,但这些蛋白质通常更难以纯化和结晶比球状蛋白8-10 。这是由以下事实卷曲螺旋结构域通常是一个较大的蛋白质的范围内发现,因此正确预测这些结构域的边界是至关重要的,以避免非结构化的或柔性的序列,其通常是有害的结晶的包含进一步加剧。

在这里,我们提出了一个概念框架,结合基于网络的计算与分析experimenta如何选择结构的研究,以及如何准备和结晶前尝试表征蛋白质样品的蛋白质片段(S):从替补l数据,通过结晶过程的初始阶段,包括帮助指导用户。我们我们的分析集中在含有大量卷曲螺旋域的两种蛋白质,蘑菇时(SHRM)和Rho激酶(岩)。这些蛋白质被选择,因为它们都包含卷曲螺旋域,并且已知以形成生物相关复杂11-16。蘑菇时和Rho激酶(岩)被预测分别含有卷曲螺旋的〜200和680个残基,多个部分,其中已被结构17-20表征。这里所描述的方法提供了一种改进的工作流程,以快速识别含蛋白卷曲螺旋的片段,就可以经得起为结晶,然而,所描述的技术可以很容易地适于大多数蛋白或蛋白复合物或修改,以结合高通量approaCHES为可用。最后,这些方法通常是廉价的,并且可以由用户在几乎所有经验水平来执行。

Protocol

注:概念框架或工作流的示意图在图1中被描述以供参考。该协议可以被分解成四个阶段:计算或基于序列的预测,蛋白质表达和纯化,生化特性和结晶。示出的实施例分析蘑菇时SD2域和/或蘑菇时摇滚络合物,但可以与任何蛋白质被利用。 1.使用建立基于Web的工具来生成卷曲螺旋域边界的计算预测收集的进化多样化序列同系物。 转到www.uniprot.org并在…

Representative Results

的图描绘在本系统中使用的工作流程示于图1,并包括三个主要阶段。序列的计算分析用于开发关于感兴趣的卷曲螺旋蛋白的结构域边界的假设。所述Shrm2 SD2域的注释分析的一个例子示于图2。在该图中,目标是确定在细胞骨架调节蘑菇时称为SD2的C末端保守结构域可能域边界。从这个分析是生成三个不同的集合假想域边界的包含跨越整个保守SD2或最…

Discussion

这里所描述的协议旨在帮助用户识别大型卷曲螺旋蛋白中域边界,以方便他们的结晶。该协议依赖于多种由计算预测和其他来源的数据的整体并入以产生一系列可能的域的边界。这些后跟一组生化实验这是快速和廉价的,并且被用来进一步缩小这些初始假设。使用这种方法,用户可以很快消除是不可取的潜在的蛋白质片段,并专注于更好的候选人更多的关注,从而提高获得晶体的前景。

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開示

The authors have nothing to disclose.

Acknowledgements

This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

Materials

BL21(DE3) Rosetta Emd Millipore 70954-3
BL21(DE3) Star ThermoFisher Scientific C601003
BL21(DE3) Codon Plus Agilent Technologies 230245
Lysozyme Spectrum Chemical Mfg Corp L3008-5GM
Ni-NTA resin Life Technologies 25216
SubtilisinA Spectrum Chemical Mfg Corp S1211-10ML
24 well Cryschem Plate Hampton research HR3-160
INTELLI-PLATE  96: Art Robbins Instruments 102-0001-03
PEG 3350 Hampton research HR2-591
PEG 8000 Hampton research HR2-515
PEG 400 Hampton research HR2-603
PEG 4000 Hampton research HR2-605
pcDNA3.1-Clover-mRuby2 Addgene 49089
Overnight Express Autoinduction System 1 Emd Millipore 71300
Lysogeny Broth powder ThermoFisher Scientific 12795027 

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記事を引用
Zalewski, J. K., Heber, S., Mo, J. H., O’Conor, K., Hildebrand, J. D., VanDemark, A. P. Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins. J. Vis. Exp. (119), e54886, doi:10.3791/54886 (2017).

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