顺磁弛豫增强，用于检测和表征固有无序蛋白质的自缔合
Summary
提出了一种应用顺磁弛豫增强核磁共振波谱来检测固有无序蛋白质中弱和瞬态分子间和分子内相互作用的方案。
Abstract
蛋白质中的内在无序蛋白质和内在无序区域构成了人类蛋白质组的重要组成部分。这些序列的高度灵活性使它们能够与不同的生物分子伙伴形成弱的、长距离的和短暂的相互作用。特异性但低亲和力的相互作用促进混杂结合，并使单个固有无序片段与多个靶位点相互作用。由于这些相互作用的瞬时性质，它们很难通过依赖蛋白质形成单一主要构象的结构生物学方法进行表征。顺磁弛豫增强核磁共振是识别和定义弱相互作用和瞬态相互作用的结构基础的有用工具。描述了使用顺磁弛豫增强来表征固有无序蛋白质与其蛋白质、核酸或其他生物分子伙伴之间形成的低人口遭遇复合物的详细协议。
Introduction
内在疾病 （ID） 描述了蛋白质 （IDP） 或蛋白质中的区域 （IDR），它们不会自发折叠成稳定的二级或三级结构，但具有生物活性。通常，IDP / IDR的功能是在生理条件下促进与生物分子的特定但可逆的相互作用1。因此，IDP 和 IDR 参与一系列细胞功能，包括多蛋白复合物的募集、组织和稳定，例如剪接体2 的组装和活性、DNA 损伤位点组件的募集和组织3、转录复合物4 或染色质重塑剂 BAF5 的募集的组织和稳定.此外，IDP存在于信号连接处，它们对不同结合伴侣的混杂性使它们能够通过细胞蛋白网络介导信息传递6。最近的工作还揭示了IDR区域通过液-液相分离过程自缔合形成生物分子冷凝物的倾向7。许多涉及ID的上述功能现在也被认为涉及冷凝水形成8的某些方面。尽管ID对于生物分子复合物组装，稳定，支架和信号转导很重要，但它们特定相互作用的原子细节很难识别，因为IDP和IDR通常不适合使用X射线晶体学或低温电子显微镜进行结构研究。
核磁共振（NMR）是研究ID的理想技术，因为它不依赖于刚性或均匀结构集合的存在，而是报告单个细胞核的直接局部环境。给定分子中原子核的共振频率或化学位移受到局部电子分布引起的弱磁场的影响，而局部电子分布又取决于键长、角度、其他原子核的接近程度、与结合伙伴的相互作用以及其他因素9.因此，每个原子核都充当独特的、特定于位点的结构探针，对其局部化学环境的变化敏感。尽管有这些优点，核磁共振是一种体型技术，观察到的化学位移是特定原子核采样的所有环境的平均值。已经开发了一系列核磁共振技术（本期中描述了其中许多技术）来恢复平均化学位移中包含的高能量、低人口生物分子构象的结构、动态和动力学信息10,11。虽然是短暂填充的，但这些状态的识别和量化对于确定功能机制的细节很重要12。例如，在IDP和IDR的情况下，构象系综可能偏向于优先采样构象，这些构象对于与生理结合伴侣形成相遇复合物是有效的。检测这些状态，以及鉴定残基特异性分子间和分子内相互作用和动力学，对于确定蛋白质功能和复合物形成的潜在结构机制非常重要。
描述了使用顺磁弛豫增强（PRE）NMR来研究对IDP / IDR介导的生物分子复合物的形成很重要的瞬态，人口稀少的状态的协议13。这种方法可用于研究瞬时蛋白质-蛋白质相互作用，例如促进α-突触核蛋白14,15 的淀粉样纤维组装或 FUS 16 的自缔合的相互作用，以及表征特定的蛋白质-蛋白质相互作用，例如信号蛋白之间的相互作用17。提出了一个自缔合IDP的例子，其中特定的分子间和分子内相互作用导致优先压缩状态以及驱动自缔合的位点特异性相互作用。
PRE起源于原子核与具有各向同性g张量的顺磁中心的磁偶极相互作用，通常以氮氧化物基团上的不成对电子的形式提供或作为顺磁性金属原子18（图1）。虽然具有各向异性g张量的原子也会产生PRE效应，但由于伪接触位移（PCS）或残余偶极耦合（RDC）产生的混杂效应，对这些系统的分析更加困难13,19。原子核和顺磁中心之间相互作用的强度取决于两者之间的<r-6>距离。这种相互作用导致核弛豫速率的增加，即使对于长程相互作用（~10-35 Å），也会导致可检测的谱线展宽，因为未成对电子的磁矩非常强20,21。如果满足以下两个条件，则可以使用 PRE 检测瞬态;（1）瞬态相互作用在核磁共振时间尺度上处于快速交换状态（观察到的化学位移是交换态的群体加权平均值）;（2）瞬态下原子核到顺磁中心的距离比主态短11。横向 PRE 表示Γ2，出于实际目的，根据包含顺磁中心和抗磁性对照的样品之间 1H 横向弛豫速率的差异计算得出。为了深入探讨快速和慢速交换制度中的PRE理论和相关伪接触位移，读者可以参考Clore及其同事的综合评论13,22。在这里，只考虑1H N-Γ2处于快速交换状态的情况，其中由于PRE的r-6依赖性，观察到的弛豫速率与顺磁中心接近原子核的距离以及在该构象中花费的时间有关。因此，不涉及接近的瞬态构象会产生较小的PRE，而较近的相互作用，即使是短暂的，也会产生较大的PRE。
对于 IDP，PRE 用于测量和区分单个分子（分子内）和单独分子（分子间）之间发生的相互作用。通过将顺磁中心连接到NMR可见（例如，15N标记）或NMR不可见（例如，天然丰度14N）蛋白上，可以确定PRE的来源（分子间或分子内）（图2）。引入半胱氨酸残基的定点诱变是将顺磁中心（自旋标记）连接到蛋白质的便捷方法23。已经提出了几种类型的分子用作自旋标记，包括金属螯合（基于EDTA）和自由基（基于氮氧化物）24。已经描述了各种氮氧化物自旋标记物，并且具有不同的半胱氨酸反应性化学物质，例如硫代磺酸盐，马来酰亚胺和碘乙酰胺25,26（图1）。标签或接头的固有灵活性对于某些分析可能是有问题的，在这些情况下，已经提出了不同的策略来限制标签的运动，例如通过添加庞大的化学基团或使用第二个接头将标签锚定到蛋白质上（两个位点附着）27，28.此外，市售标签可能含有非对映异构体蛋白，但通常这不会有助于观察到的PRE29。描述了通过马来酰亚胺化学连接到游离半胱氨酸上的3-Maleimido-PROXY的使用，因为它易于获得，具有成本效益，不可逆，并且还原剂三（2-羧乙基）膦（TCEP）可以在整个标记反应中维持在溶液中。由于3-Maleimido-PROXYL具有各向同性的g-张量，因此不会诱导PCS或RDC，并且相同的化学位移分配可用于顺磁性和抗磁性样品13。
1HN-T 2 使用两个时间点策略（T a，T b）进行测量，该策略先前已被证明与收集由 8 到 12 个时间点30 组成的完整进化序列一样准确。第一个时间点（T a）设置为尽可能接近零，第二个时间点的最佳长度取决于给定样品的最大预期PRE的大小，可以由下式估计：Tb ~ 1.15/（R 2，dia + Γ 2），其中R 2，dia表示抗磁性样品13的R 2。如果最大 PREs 的大小未知，将 T b 设置为 ~ 蛋白质 1 H T 2 的 1倍是一个很好的初始估计，并通过调整 T2 来进一步优化以改善信噪比。这种两点测量策略显著减少了测量PRE所需的实验时间，并允许有更多的时间进行更多的信号平均，特别是因为使用相对稀释的样品来最小化分子之间非特异性接触的影响。基于HSQC的脉冲序列用于测量1H N-T2，并在其他地方进行了详细描述30。为了提高灵敏度，前向和后向INEPT转移的硬脉冲可以用整形脉冲代替;或者，该序列容易地转换为基于TROSY的读出31。由于IDP通常具有更长的横向弛豫率，导致线宽（由于固有的紊乱）比类似大小的球状蛋白更窄，因此间接维度的长采集时间可用于提高光谱分辨率并减轻IDP固有的化学位移色散限制。
PRE是研究蛋白质-蛋白质和蛋白质-核酸相互作用的有用工具，特别是瞬时或人口稀少的相互作用。提供了用于制备适用于测量PRE的NMR样品的详细方案，包括蛋白质纯化，定点自旋标记，设置和校准脉冲程序，处理和解释NMR数据的步骤。自始至终都注意到了可能影响数据质量和实验结果的重要实验考虑因素，包括样品浓度、自旋标记的选择和顺磁性成分的去除。
Protocol
Representative Results
Discussion
已经提出了一种使用PRE表征固有无序蛋白质和各种结合伴侣之间在低群体中存在的瞬时相互作用的方法。在所示的示例中，蛋白质是自缔合的，因此PRE可能来自分子间和分子内相互作用的组合。该方法很容易扩展到异质样品，其中可以表征两种不同蛋白质之间的相互作用。通过将自旋标记放置在蛋白质内的不同位置，可以获得有关蛋白质不同区域如何相互作用的补充信息。此外，通过在NMR活性（<…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢应金发博士和克里斯汀·卡诺博士的有益讨论和技术援助。DSL是圣鲍德里克学者，并感谢圣鲍德里克基金会（634706）的支持。这项工作得到了韦尔奇基金会（AQ-2001-20190330）对DSL的部分支持，Max and Minnie Tomerlin Voelcker基金（Voelcker基金会青年研究员资助DSL），UTHSA启动基金DSL，以及Greehey儿童健康研究生奖学金CNJ。这项工作基于在结构生物学核心设施中进行的研究，这是德克萨斯大学圣安东尼奥健康科学中心机构研究核心的一部分，由研究副总统办公室和梅斯癌症中心药物发现和结构生物学共享资源（NIH P30 CA054174）支持。
Materials
| 0.45 µm and 0.22 µm syringe filters | Millipore Sigma | SLHVM33RS SLGVR33RS |
Filter lysate before first purification step and before size exclusion chromatography. |
| 100 mm Petri Dish | Fisher | FB0875713 | Agar plates for bacterial transformation. |
| 14N Ammonium chloride | Sigma Aldrich | 576794 | Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein |
| 15N Ammonium chloride | Sigma Aldrich | 299251 | Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein |
| 3 L Fernbach baffled flask | Corning | 431523 | Bacterial expression culture |
| 3-Maleimido-Proxyl | Sigma Aldrich | 253375 | Nitroxide spin label |
| 50 mL conical centrifuge tubes | Thermo Fisher | 14-432-22 | Solution/protein storage |
| Amicon centrifugal filter | Millipore Sigma | UFC900308 | Protein concentration |
| Ampicillan | Sigma Aldrich | A5354 | Antibiotic for a selective marker, exact choice depends on the expression construct plasmid |
| Analytical balance | Oahus | 30061978 | Explorer Pro, for weighing reagents |
| Ascorbic acid | Sigma Aldrich | AX1775 | Reduces nitroxide spin label |
| Autoclave | Sterilize glassware and culture media | ||
| Calcium chloride | Sigma Aldrich | C4901 | M9 media component |
| Centrifuge bottles | Thermo Fisher | 010-1459 | Harvest E. coli cells after recombinant protein expression |
| Centrifuge, hand-crank | Thomas Scientific | 0241C68 | Boekel hand-driven, low-speed centrifuge with 15 mL buckets that can accommodate NMR tubes |
| Chelex 100 | Sigma Aldrich | C7901 | Remove contaminating paramagnetic compounds from buffer solutions |
| Computer workstation | Linux or Mac OS compatable with NMR data processing and analysis software packages such as NMRPipe and Sparky | ||
| Deuterium oxide | Sigma Aldrich | 151882 | Needed for NMR lock signal |
| Dextrose | Sigma Aldrich | D9434 | M9 media component |
| Dibasic Sodium Phosphate | Sigma Aldrich | S5136 | M9 media component |
| Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid) | Thermo Fisher | 22582 | Quantification of free cystiene residues |
| High speed centrifuge tubes | Thermo Fisher | 3114-0050 | Used to clear bacterial lysate. |
| High-field NMR instrument (600 – 800 MHz) | Bruker | Equiped with a multichannel cryogenic probe and temperature control | |
| IMAC column, HisTrap FF | Cytvia | 17528601 | Initial fractionation of crude bacterial lysate |
| Isopropyl B-D-thiogalactoside (IPTG) | Sigma Aldrich | I6758 | Induces protein expression for genes under control of lac operator |
| LB agar | Thermo Fisher | 22700025 | Items are used for transforming E. coli to express protein of interest, substitions for any of these items with like products is acceptable. |
| LB broth | Thermo Fisher | 12780052 | |
| Low-pressure chromatography system | Bio-Rad | 7318300 | BioRad BioLogic is used for low-pressure chomatograph such as running IMAC columns |
| Magnesium sulfate | Sigma Aldrich | M7506 | M9 media component |
| Medium pressure chromatography system | Bio-Rad | 7880007 | BioRad NGC equipped with a multi-wavelength detector, pH and conductivity monitors, and automatic fraction collector |
| MEM vitamin solution | Sigma Aldrich | M6895 | M9 media component |
| Microfluidizer | Avestin | EmulsiFlex-C3 | Provides rapid and efficient bacterial cell lysis |
| Micropipettes | Thermo Fisher | Calibrated set of micropippetters with properly fitting disposable tips (available from multiple manufacturers e.g. Eppendorf) | |
| Monobasic potassium phosphate | Sigma Aldrich | 1551139 | M9 media component |
| NMR pipettes | Sigma Aldrich | 255688 | To remove sample from NMR tube |
| NMR sample tube | NewEra | NE-SL5 | Suitable for high-field NMR spectrometers |
| Preparative Centrifuge | Beckman Coulter | Avanti J-HC | Harvest E. coli cells after recombinant protein expression |
| Round bottom polystyrene centrifuge tubes | Corning | 352057 | Clear bacterial lysate |
| Shaking incubator | Eppendorf | S44I200005 | Temperature controlled growth of E. coli starter and expression cultures |
| Sodium chloride | Sigma Aldrich | S5886 | M9 media component |
| Sonicating water bath and vacuum source | Thomas Scientific | Used to degas buffer solutions | |
| Sonicator | Thermo Fisher | FB505110 | Used for bacterial cell lysis or shearing bacterial DNA |
| Spectrophotometer | Implen | OD600 Diluphotometer | Monitor growth of E.coli protein expression cultures |
| Superdex 200 16/600 size exculsion colum | Cytvia | 28989333 | Final protein purification step |
| Topspin software, version 3.2 or later | Bruker | Operating software for the NMR instrument | |
| Transformation competent E. coli cells | Thermo Fisher | C600003 | One Shot BL21 Star (DE3) chemically competent E. coli, other strains may be compatable |
| Tris(2-carboxyethyl)phosphine (TCEP) | ThermoFisher | 20490 | Reducing agent compatable with some sulfhydryl-reactive conjugations |
| UV-Vis spectrophotometer | Implen | NP80 | Measure protein concentration. |
| Water bath, temperature controlled | ThermoFisher | FSGPD25 | For heat shock step of bacterial transformation |
| Yeast extract | Sigma Aldrich | Y1625 | For supplementing M9 media if required |
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