我们承认 OPPF-英国在克隆技术方面的帮助, 哈里斯为 SEC 购物中心和光束 I03 和 I04 在钻石光源的科学家。

注意: 信号肽被删除的编码序列在标准的融合协议后的 pOPINF 向量中被克隆; 本机蛋白在大肠杆菌中被表达为他的标记融合 大肠埃希氏大肠杆菌 BL21 罗塞塔细胞 28 , 29 . 硒标记的变体按照制造商 30 的标准方法表示。重组的收缩蛋白被纯化为先前描述的 31 , 32 。
 1. 生化特性 <ol> <li> 热转移检测 <ol> <li> 准备48个具有 pH 范围的缓冲液, 从4.0 到 9.5, 氯化钠浓度从0到0.5 米, 并分配40和 #181; 在96井板中描述 表 1 33 . </li> <li> 添加到每个井5和 #181; 在 1-2 毫克/毫升浓度的情况下, 该方案的 L. </li>
		<li> 添加到每个井5和 #181; L 20x 和 #160; 蛋白质的荧光染色 (请参阅 材料表 ). </li>
		<li> 将解决方案由移混合, 用透明的粘合剂膜密封该板, 室温下在 112 x g 处旋转2分钟. </li>
		<li> 在 real-time 机上运行实验: 设置3和 #176 的温度斜坡; c/分钟从4到99和 #176; c 与十年代保持时间, 并且读荧光每0.5 和 #176; c 与励磁在483毫微米和放射在568毫微米. </li>
		<li> 分析每个缓冲条件的荧光发射曲线, 确定熔化温度 (T m ) 作为导数的最小值. 
		注意: 这个过程提供了最好的缓冲液, 以帮助改善蛋白质的稳定性。根据具有最高 T m 的缓冲条件选择 pH 值范围和盐浓度, 并添加 2.5% (v/v) 的甘油和0.5 毫米的三 (2-乙) 膦 (TCEP), 以定义以下步骤的缓冲液 (秒缓冲). </li>
	</ol> </li> <li> 加工和分析秒 <ol> <li> 使用脱过滤水的包装凝胶过滤柱进行两柱体洗涤 (用宽范围的分子量 24 mL 柱). </li>
		<li> 将柱形连接到光散射检测器上, 并平衡从热位移检测中确定的秒缓冲柱. </li>
		<li> 注入100和 #181; 在5毫克/毫升的纯化的平衡秒列, 并流一列的 sec 缓冲区的体积. </li>
		<li> 检查洗脱色谱是否为单个或多个峰值, 并使用分析软件检测散射数据, 获取所有物种的摩尔质量和多指数. 
		注: 对于每一个聚物种的峰值, 在质量/摩尔质量加权的摩尔质量 (兆瓦/锰) 上, 对多指数进行检验是有益的。接近1的多值表示分散峰值. 
		注意: 一旦所有的齐聚物种被定义, 研究它们对蛋白质浓度的依赖性是有用的, 因为较高的浓度可能有利于更大的齐聚物形成. </li>
		<li> 执行多个分析秒运行; 注入100和 #181; 向平衡凝胶过滤塔 (0.1-10 毫克/毫升) 增加浓度 (使用宽范围的分子量 5 ml 柱), 每一次流一列的秒缓冲液. </li>
		<li> 检查洗脱色谱的单个或多个峰值, 并分析与不同起始蛋白质浓度对应的 280 nm 曲线的吸光度强度。如果不同峰的相对强度保持不变, 则不存在聚物种之间的改制。不同浓度的相对强度的变化表明, 不同聚物种之间的转换依赖于蛋白质浓度. 
		注意: 这个表征步骤是定义哪些物种更适合于结晶的基础。事实上, 不同的分散物种更容易结晶, 如果它们是在一个稳定的聚状态在一个明确的浓度. </li>
	</ol> </li> </ol> 2。蛋白质制备与结晶 <ol> <li> 准备秒 <ol> <li> 在用脱过滤水进行一柱式容积洗涤后, 平衡用秒缓冲剂制备的包装凝胶过滤柱 (宽范围分子量120毫升列). </li>
		<li> 向平衡凝胶过滤柱注入 1-5 毫升的纯化的收缩剂, 并将一柱流量的秒数缓冲. </li>
		<li> 以1毫升的分数收集列流. </li>
		<li> 将与单个单分散峰值相对应的分数汇集在一起; 在15毫升离心过滤器中旋转1500克的样品, 并将浓度量化, 直到达到所需浓度 (通常为 SP0092 的 50-100 毫克/毫升). </li>
	</ol> </li> <li> 结晶 <ol> <li> 使用制造商描述的预结晶试验确定结晶试验的最佳浓度范围: <ol> <li> 0.5-1.0 毫升每四结晶试验解决方案在一个不同的油藏24井下落结晶板. </li>
			<li> 将1和 #181 的蛋白质溶液与1和 #181 的溶出液混合, 在室温下孵育不少于1小时 (在夜间孵化后获得更可靠的结果). </li>
			<li> 使用10x 和 #160 检查每个浓度的跌落质量; 放大显微镜 
			注: 测试至少三种不同的蛋白质浓度: (I) 大部分残留的水滴清楚表明, 蛋白质太稀, 无法结晶;(二) 在大多数下落中出现大量沉淀, 意味着浓度过高;和 (III) 平衡的发生, 无论是明确的下降和沉淀 (更好的, 如果光) 是一个很好的迹象表明, 测试浓度有利于结晶. </li>
		</ol> </li> <li> 准备96个井下的下落结晶板; 分配100和 #181; 在每个油藏中不同的商业结晶溶液 L 34 , 35 , 36 , 37 . </li> <li> 使用晶化机器人系统在所有的结晶滴井中, 在先前定义的浓度 (步骤 1.1.6) 中分配 100 nL 蛋白质样本. </li>
		<li> 将 100 nL 不同的油藏溶液分配给相应的结晶滴井, 与步骤2.2.3 中的蛋白质混合。密封的结晶板, 以避免蒸发, 使平衡的结晶下降与水库. </li>
		<li> 每 1-2 天, 每个星期晚些时候) 使用10x 和 #160;(至少) 放大显微镜来评估晶体的形成和生长. 
		注: 用于蛋白质结晶的自动成像系统可用于水滴可视化和捕获. </li>
	</ol> </li> </ol> p 类 = "jove_title ">> 3。晶体特性和 x 射线数据收集 <ol> <li> 晶体安装 <ol> <li> 准备剂溶液, 方法是将甘油 (最终浓度) 的 25% (v/v) 添加到结晶条件 (从而在初始状态下替换25% 的水混合) 38 . </li>
		<li> 用液氮填充泡沫杜瓦, 并将单冰球的样品箱部分放入杜瓦瓶中。让它冷却到液氮温度. </li>
		<li> 切割和从结晶板上取下密封胶带, 在晶体形成的下落处. </li>
		<li> 将一滴1和 #181; 剂解决方案放置在靠近目标下落的 coverslide 上. </li>
		<li> 将所选的晶体从原始下落转移到剂溶液下落使用在磁性棒上安装的脊柱标准底座上的尼龙冷冻环 39 , 40 . </li>
		<li> 快速将晶体从剂滴转移到液氮, 将该回路放入单冰球样品架的第一个空位置. </li>
		<li> 重复 (从密封胶带切割步骤), 直到所有所需的晶体收获和存储在单冰球样品持有人. </li>
		<li> 使用冰球棒将单冰球基地放在单冰球上, 然后将冰球带到光束 (在液氮条件下). 
		注意: 温度极低! </li>
		<li> 使用冷冻钳和冰球杜瓦加载工具加载的单冰球 (s) 到光束样品更换机器人杜瓦瓶。单冰球样品持有人将分离从基地盖子离开样本循环持有人直立在样品机器人杜瓦, 从而暴露在液氮和可访问的样品更换机器人. </li>
	</ol> </li> <li> 晶体特性 注意: 可以使用实验室 x 射线源或同步辐射 (SR) x 射线源对收获的晶体进行衍射质量筛选。高分子晶体学 (MX) 光束提供了一种可调谐的能量源来利用反常衍射进行结构解。在下面的部分中, 提出了一系列的工作指示, 与实验能力的钻石光源 mx 光束, 但这些准则也可以适应 mx 光束在其他加速器世界各地。
	<ol> <li> 作为第一步, 确认或识别晶体中的反常散射是很有用的。通过测量晶体的 x 射线荧光光谱可以方便地实现这一目标。首先确保光束的 X 射线能量设置得足够高, 以激发大分子晶体 (14 凯文或更高) 通常预期的所有元素. </li>
		<li> 使用光束控制软件选择由采样转换器安装的样品; 回路和 #160; 将自动集中在 X 射线束上, 并可手动确认晶体定心, #160; 使用光束控制软件. </li>
		<li> 使用光束控制软件记录 X 射线荧光光谱: 用户选择曝光时间并启动测量 (荧光/荧光光谱设置, 和 #8594; 运行, 图 2A )。光束控制软件自动定位 x 射线荧光探测器到位, 并确定最小入射 x 射线通量, 以获得在荧光探测器上的可读信号。然后以自动的方式分析获得的频谱, 并将发射峰安装在自然发生的生物元素上。这些例程可在我们公司的光束控制软件图形用户界面 (GUI)-GDA (www.opengda.org) 中获得, 在整个欧洲加速器 41 , 42 . </li>
		<li> 如果确定了可用于反常衍射实验的合适元素, 则执行 X 射线吸收边缘能量扫描, 以确定从晶体中收集异常衍射数据的最佳波长: 在光束上控制软件, 选择元素并启动扫描 (荧光/荧光扫描设置, 和 #8594; Atom 名称, 和 #8594; 运行, 图 2B ). 
		注: 对于一个单一的反常衍射实验, 吸收边缘的峰值被用来最大化的反常散射。进行多波长反常衍射实验的实验考虑以前是详细的 43 . </li>
		<li> 要确定晶体单元的单元参数、对称性和衍射极限, 请在45和 #176 测量三 x 射线衍射图样; 使用振荡方法的间隔 (数据收集/筛选和 #160; #8594; 运行当前, 图 2C )。光束控制软件为用户提供了一个振荡角度和曝光时间的选择, 并能够设定 X 射线束的透射率。对于标准的晶体筛查, 建议了0.5 和 #176 的振荡角; 0.5 秒的曝光时间和 5% X 射线束传输。所测得的衍射图像由埃德娜管道自动分析, 并返回一组完整的数据集 22 的集合策略. </li>
	</ol> </li> <li> X 射线数据收集 注意: 用户可以选择在标准振荡模式或逆波束模式下收集数据, 这使弗里德尔的队友能够在时间和 X 射线剂量之间进行记录, 并具有近似相同的吸收行为允许更精确地测量反常的差异。后者是有用的, 特别是如果预期小的反常差异和/或样品是辐射敏感的, 当进行一个反常的衍射实验。有关使用的最佳数据收集策略的注意事项已被全面审阅 22 , 23 , 44 , 45 , 46 , 47 . <ol> <li> 使用光束控制软件导入数据收集参数, 该策略计划将提供: i. #969 的起始位置;-旋转轴 ii。#969 的振荡宽度;-每个衍射图像的轴旋转角度 iii。每个衍射图像的曝光时间 iv。完整数据集的图像数 (间接定义总计和 #969;-整个数据采集的轴旋转角度) v. x 射线束衰减的百分比, 以避免过度和辐射损伤 注意: 在我们的机构,运行数据收集时, 自动处理收集的数据的软件管道已经建立好了: (i) 通过 xia2 管道减少 (索引、集成和缩放) 的衍射数据, 从而为用户生成反射 mtz 文件作为分阶段和结构解决方案/细化的输入 48 。(ii) 在数据分析过程中检测到一个重要的反常信号时, 使用 SHELX 的第一个快速自动结构解决方案管道 (fast_ep) 尝试通过实验分期解决重原子下部, 提供了相控电子密度图在可行的情况下。第二个更全面的结构解决方案管道自动承担解决和建立结构的尝试重新使用独立软件套件 49 , 50 , 51 , 52 ;在成功的情况下, 用户将得到一个初始模型和电子密度图。这为用户提供了根据所选择的晶体软件套件完成细化和验证模型的基础. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

在本文中, 我们描述和验证一个综合协议的生物化学和结构表征的碳水化合物 SBPs, 特别强调蛋白质从S. 肺炎链球菌。然而, 这可以作为一个标准的程序来分析其他 SBPs 从不同的有机体, 甚至其他无关的可溶性蛋白质。
该协议的第一部分着重于提供蛋白质稳定性和第四纪结构的生物化学信息, 可用于制备蛋白质样品进行结晶。在热转移分析部分, 我们只描述 pH 和氯化钠浓度的变化, 以保持这个过程的一般性质。尽管如此, 许多其他的缓冲条件可以用类似的方法进行测试, 例如, 包括任何用作稳定添加剂的化合物: 特别是与特定的收缩压结合的实际配体, 在增加 Tm数摄氏度31。在某些情况下, 由于低信号或高荧光背景而导致的变性曲线的定义很差, 这是由蛋白质聚集或部分展开引起的。为了避免这种变化, 一种蛋白质: 可以进行染料滴定来优化不清楚的变性曲线。如果没有得到改善, 筛选各种添加剂, 可以改善蛋白质的稳定性是建议, 和合适的屏幕已经被描述为54。
通常, 大多数 SBPs 是单体在他们的自然环境, 但如图所示, multimerization 可以发生在较高浓度使用的结晶实验, 从而由加工和 SEC 提供的齐聚行为表征重要的是要评估最有利稳定的单分散齐聚状态的结晶。然而, 很难预测在结晶条件中所含的不同化学物质对蛋白质齐聚行为的影响。如果 SEC 和加工检查显示了蛋白质样本的广泛聚集, 我们将建议以下的方法来减少发生这种情况的可能性: 使用新鲜的蛋白质样本 (不冻融) 和扩大热稳定性分析转移化验, 测试可能的添加剂和温和的洗涤剂作为最后的资源, 以尽量减少聚合。本文提出了利用高通量稀疏矩阵商业结晶筛选的基本准则, 以保持该协议的一般性质。然而, 获得高分辨率 X 射线衍射蛋白晶体可能需要迭代微调, 以优化结晶条件的沉淀浓度, pH 值, 添加化学添加剂, 不同的温度,其他影响晶体下落与储层平衡动力学的因素16,17。
该协议的第二部分描述了蛋白质晶体的表征, 以确定 X 射线衍射数据收集的最佳策略, 并特别着重于采集异常数据进行 SAD 分期。即使 SBPs 保持一个类似的一般架构 (有许多沉积的3D 结构可能是可用的作为启动模型), 这些蛋白质的分期的分子置换方法并不总是直接的, 因为变化的二级结构元素和这些蛋白质的内在灵活性。因此, 我们提出可悲的方法, 并强调, 这些蛋白质可能已经有内在的约束金属或事实上的非特异的结合金属从结晶缓冲条件, 这可以提供一系列反常的衍射元素作为我们的常规协议中的标准步骤。
最后, 本协议定义了一个标准的指导工作流程, 以便能够详细描述 SBPs 的生物化学和结构特征, 以提高结构确定成功率, 并加快一般 SBPs 的结构特征。

S. 肺炎球菌) 是一种 gram-positive 细菌, 居住在人类呼吸道上部的症状, 能够迁移到正常的无菌龛位, 导致中耳炎、肺炎、败血症、败血症和脑膜炎1,2。此外, 肺炎球菌感染是导致社区获得性肺炎的主要原因, 它在全球范围内造成了临床和经济负担3,4。抗生素耐药性菌株的S. 肺炎病毒已遍布全球, 虽然七价和十三价的肺炎球菌蛋白共轭疫苗有助于降低抗菌素耐药性率, 取代菌株疫苗的使用已经出现, 并导致对肺炎球菌病新治疗方法的研究的需求增加5,6,7,8。
肺炎球菌依赖于从主机输入的糖类作为碳源9,10;事实上, 它致力于30% 的进口机械的运输32种不同的碳水化合物11,12,13。这些进口商包括至少八 ABC 运输商13。在 ABC 转运器中, 胞外 SBPs 在确定配体特异性方面起着根本性的作用, 并将其呈递给细胞膜上的整体载体以供细胞吸收。SBPs 代表了新疫苗和抗菌素设计的有效目标, 因为它们是表面蛋白及其在细胞过程中的重要作用。
靶蛋白的表征和结构特征的详细描述, 如配体袋和域的灵活性, 为结构药物设计提供了一个有用的工具14,15。x-射线结晶学是选择的方法为蛋白质的结构描述在接近原子决议, 但结晶过程是不可预知的, 费时, 并且不总成功。系统的方法提高了成功率, 重要的因素是样品的质量和稳定性。结晶的成功率受蛋白质化学性质和样品制备方法的影响。它们的作用可以通过生物化学特性16,17进行评估和通知。
一个进一步复杂化为结构设计是晶体阶段问题, 必须解决。随着更多的蛋白质结构变得可用, 许多结构可以通过分子置换的方法来解决, 这需要一个同源结构18。由于 SBPs 提出了一个灵活的领域结构, 分子置换也可能证明挑战19。如果一个结构模型是足够相似的目标蛋白是不可用的, 一些技术可以用来获得实验阶段的20。其中, 单波长反常色散 (SAD) 方法已成为主要技术, 已被广泛用于解决21的相位问题。使用 SAD 方法进一步改进了硬件和软件, 以及数据收集策略, 以便能够检测和使用微弱的异常信号, 以便逐步进行22,23, 24. 此外, 直接解决大分子结构的方法的进展, 这在过去需要衍射数据的原子分辨率, 现在可以利用结合, 例如, 立体知识, 在程序中实施ARCIMOLDO25。泰勒26给出了在晶体学中解决相位问题的方法的一个有用的综述。
在这里, 我们提出了一个合理化的协议的表征碳水化合物运输收缩, SP0092 的S. 肺炎, 集成生化和结构技术 (图 1)。这一 step-by 的协议提供了一个有用的例子测试的策略, 以提高结构研究的成功率, SBPs 一般, 这是在所有王国生活中发现。特别是, 该协议强调了在快速有效的方法中确定蛋白质在溶液中最稳定的聚状态的重要性, 并允许对最佳品种进行结晶实验的鉴定。虽然在蛋白质数据库27中报告了超过500个结构化的收缩组织, 但是分子置换可能具有挑战性, 这是由于两个α/β域的固有弹性性质, 它们由铰链区域19连接。因此, 该协议的第二部分描述了使用 sad 的方法逐步从束缚金属离子, 这是常见的 SBPs, 以及纳入硒和使用硒 (Se) 在 SAD 分期。

<table>
	<tbody>
		<tr>
			<td>SelenoMethionine Medium Complete</td>
			<td>Molecular Dimensions</td>
			<td>MD12-500</td>
			<td>Based on a synthetic M9 minimal media supplemented with glucose, vitamins and amino acids with the exception of L-methionine. Other equivalent products are commercially available by other companies.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-Well plate</td>
			<td>Applied Biosystems</td>
			<td>4306737</td>
			<td>The Applied Biosystems MicroAmp Optical 96-Well Reaction Plate with Barcode is optimized to provide unmatched temperature accuracy and uniformity for fast, efficient PCR amplification. This plate, constructed from a single rigid piece of polypropylene in a 96-well format, is compatible with Applied Biosystems&#174; 96-Well Real-Time PCR systems and thermal cyclers.</td>
		</tr>
		<tr>
			<td>SYPRO Orange</td>
			<td>Molecular Probes</td>
			<td>S6651</td>
			<td>SYPRO Orange Protein Gel Stain is a sensitive, ready-to-use fluorescent stain for proteins in 1D gels. Quite universal and well-established protein dye for hydrophobic regions.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive film</td>
			<td>Applied Biosystems</td>
			<td>4311971</td>
			<td>The Applied Biosystems MicroAmp Optical Adhesive Film reduces the chance of well-to-well contamination and sample evaporation when applied to a microplate. It is ideal for optical measurement, because it gives low background.</td>
		</tr>
		<tr>
			<td>7500 Fast Real-time PCR System</td>
			<td>Applied Biosystems</td>
			<td>4362143</td>
			<td>The Applied Biosystems 7500 Fast Real-Time PCR System enables standard 96-well format high speed thermal cycling, significantly reducing your run time for quantitative real-time PCR applications, delivering results in about 30 minutes. The Upgrade Kit is available to upgrade a standard 7500 Real-Time PCR System to the Fast Configuration via a field service installation. Other RT-PCR machine can be used. Data export not easy for the old data analysis software.</td>
		</tr>
		<tr>
			<td>Superdex 200 increase 10/300 GL column</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td>Superdex 200 Increase 10/300 GL is a versatile, prepacked column for size exclusion chromatography in small-scale (mg) preparative purification as well as for characterization and analysis of proteins with molecular weights between 10 000 and 600 000, such as antibodies. Optimal separation ideal for high resolution biophysical techniques.</td>
		</tr>
		<tr>
			<td>DAWN HELEOS II</td>
			<td>Wyatt</td>
			<td></td>
			<td>DAWN HELEOS II is the premier Multi-Angle static Light Scattering (MALS) detector for absolute characterization of the molar mass and size of macromolecules and nanoparticles in solution. The DAWN offers the highest sensitivity, the widest range of molecular weight, size and concentration, and the largest selection of configurations and optional modules for enhanced capabiliites. Other MALS detecting systems from other companies apart from Wyatt have not been tested, so no additional feedback can be provided.</td>
		</tr>
		<tr>
			<td>Superdex 200 5/150 GL</td>
			<td>GE Healthcare</td>
			<td>28906561</td>
			<td>Superdex 200 are prepacked size exclusion chromatography columns for high-resolution small-scale preparative and analytical separations of biomolecules. Superdex 200 has a separation range for molecules with molecular weights between 10 000 and 600 000. The peak separation is not as optimal as for the &#34;increase&#34; version but this model is ideal for the standard day by day use.</td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 200 pg</td>
			<td>GE Healthcare</td>
			<td>28989335</td>
			<td>HiLoad 16/600 Superdex 200 prep grade are prepacked XK columns designed for high-resolution preparative gel filtration chromatography.</td>
		</tr>
		<tr>
			<td>24 Well &#34;Big&#34; Sitting Drop Crystallization Plate</td>
			<td>MiTeGen</td>
			<td>XQ-P-24S-A</td>
			<td>The 24 well &#34;big&#34; crystallization plate is used mainly for protein crystal screening by sitting drop vapor diffusion techniques, and for crystallization condition optimization. It has quite large reservoir well and sample container, which favor manual handling and big sized protein crystal growth. In addition, its flat surfaces are easily to be sealed by transparent tape or cover slips.</td>
		</tr>
		<tr>
			<td>PCT Pre-Crystallization Test</td>
			<td>Hampton</td>
			<td>HR2-140</td>
			<td>The PCT Pre-Crystallization Test is used to determine the appropriate protein concentration for crystallization screening.</td>
		</tr>
		<tr>
			<td>96 Well CrystalQuick</td>
			<td>Greiner bio-one</td>
			<td>6098xx</td>
			<td>Square-well plates have three crystallization wells per reservoir, making it possible to test 288 samples per plate. Generally used only for the inital screening because their squared edge make crystals fishing difficult.</td>
		</tr>
		<tr>
			<td>Uni-Puck</td>
			<td>Molecular Dimensions</td>
			<td>MD7-601</td>
			<td>The Universal V1-Puck (Uni-puck) is a sample pin storage and shipping container for use with the majority of automated sample mounting systems worldwide - includes the ACTOR, SAM and CATS systems amongst others (Diamond, Soleil, SPring-8, Photon Factory, CLSI and across the USA)</td>
		</tr>
		<tr>
			<td>Standard Foam Dewar</td>
			<td>Molecular Dimensions</td>
			<td>MD7-35</td>
			<td>5.7&#34; diameter by 2.8&#34; deep. 800mL capacity.</td>
		</tr>
		<tr>
			<td>Mounted CryoLoop - 20 micron</td>
			<td>Hampton</td>
			<td>HR4-955</td>
			<td>Mounted CryoLoops with 20 micron diameter nylon. These nylon loops are bonded to hollow, stainless steel MicroTubes&#8482; that are used to mount, freeze, and secure the crystal during cryocrystallographic procedures and X-ray data collection. Different sizes exist and they can be adapted in lenght. They are quite versatile tools.</td>
		</tr>
		<tr>
			<td>CryoWand</td>
			<td>Molecular Dimensions</td>
			<td>MD7-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Puck dewar loading tool</td>
			<td>Molecular Dimensions</td>
			<td>MD7-607</td>
			<td>This tool is used to separate uni-pucks to load them into the robot dewar. It consists of two pieces: a Teflon tube part and a metal rod part.</td>
		</tr>
	</tbody>
</table>

biochemical and structural characterization of the carbohydrate transport substrate-binding-protein sp0092

为防止多重耐药性菌株的迅速增加, 必须研制新的抗菌素和疫苗, 以防止肺炎链球菌的。碳水化合物基质结合蛋白 (SBPs) 是发展蛋白质疫苗和新抗菌素的可行目标, 因为它们的胞外定位和对肺炎球菌代谢的碳水化合物进口的中心地位,分别.这里描述的是一个合理化的综合协议, 对 SP0092 进行全面的表征, 可以扩展到其他碳水化合物 SBPs 从肺炎球菌和其他细菌。这一过程可以帮助结构设计这类蛋白质的抑制剂。本文的第一部分介绍了热位移法、多角度光散射 (加工) 和尺寸排斥色谱 (SEC) 的生化分析协议, 该方法优化了样品的稳定性和均匀性, 以结晶试验, 从而提高了成功的几率。本程序的第二部分描述了使用可调谐波长的反常衍射同步光束, 以及可用于解析结晶蛋白的数据的数据收集协议。结构.

这一综合协议已被证明是成功的四 (两个出版和两个未发表的结构) 的六碳水化合物结合蛋白目标从肺炎球菌分析到日期32,53。在本节中, 我们介绍了 SP0092 的生物化学和结构特征, 作为指导 SBPs 结构研究的典型结果。
在 SP0092 已被表达和纯化为先前定义的32后, 用热漂移法分析了纯化蛋白的缓冲稳定性: SP0092 在 pH 值6.5 和氯化钠的存在下, 在 0-0.2 m 中呈现增加的 Tm浓度范围 (图 3A)。因此, 以下步骤的缓冲解决方案被定义为: 0.02 m MES pH 6.5、0.2 m 氯化钠、2.5% (v/v) 甘油、0.5 mM TCEP。SP0092 的不同齐聚态的绝对摩尔质量是由加工耦合到 SEC 测量187.2、140.8、97.0 和 49.4 kDa 的分子量, 分别对应于 tetrameric、三、二和单体物种 (图 3B)。对不同蛋白稀释的 SEC 剖面的分析表明, 齐聚物是由增加的蛋白质浓度引发的, 这表明更大的寡聚物在较高的浓度下比通常使用的更稳定。结晶.事实上, 被纯化的较大的聚物种的结晶试验, 成功地产生了蛋白质晶体, 而单体物种没有。
用 X 射线衍射法表征了 SP0092 的天然和硒-蛋氨酸标记形式的优化晶体。这些晶体中的 X 射线荧光的测量结果显示, 在两种情况下, 锌与蛋白质结合的发射峰, 而硒只被发现的硒-蛋氨酸晶体的预期。随后, 在硒和锌边缘进行 x 射线吸收扫描, 提供了直接的实验数据, 将入射 x 射线波长调整到晶体中锌或硒的各自 x 射线吸收边缘, 使异常信号达到最大。可从结果数据中获得 (图 4A-B)。
在低传输测量三衍射模式后, 利用埃德娜 (图 4C) 所建议的数据收集策略获得了一个完整的异常数据集。异常信号的出现触发自动分阶段管道在光束, 以确定结构, 并根据最初的实验阶段得出, 产生初始地图和模型, 然后可以提炼和验证 (图4D).
总之, 技术的潜在缺陷主要集中在晶体的可用性和质量上。优化的缓冲条件, 以提高蛋白质的稳定性, 以及确定最合适的聚状态的蛋白质使用, 当多个齐聚物被确定在溶液中, 可以减少失效的风险在结晶阶段.利用早期发现的束缚金属离子, 可以加快结构的解决, 避免在分子置换方法失败时不必要的硒标记蛋白的产生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56294/56294fig1.jpg"> 
图1。碳水化合物 SBPs 的生物化学和结构表征工作流程图.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56294/56294fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56294/56294fig2.jpg"> 
图2。GDA 中的晶体特性.(A) GDA 光束控制软件的 X 射线荧光控制标签的屏幕截图。(B) GDA 中 X 射线能量边缘扫描控制卡的屏幕截图。(C) GDA 中的数据收集水晶筛选选项卡的屏幕快照。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56294/56294fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56294/56294fig3.jpg"> 
图3。SP0092 的生物化学特性39-491.(A) 3 维曲面图, 它将 SP009239-491的熔化温度绘制为缓冲液的 pH 和氯化钠浓度的函数。(B) SEC 和加工结果为 SP009239-491。在 280 nm 的吸收以蓝色显示。不同齐聚状态的摩尔质量以红色显示。面板 (B) 已从32中修改。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56294/56294fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56294/56294fig4.jpg"> 
图4。SP009239-491的结构特征.(A) 和 (B) X 射线吸收能量扫描在 SP009239-491晶体的锌和硒边缘。从含锌和硒的晶体中测定的荧光值分别显示为蓝色和青色, 计算出的 f "和 f" 异常散射分数为绿色和红色。(C) 从 SP009239-491晶体中收集的 X 射线衍射图案的示例。(D) SP009239-491二聚体结构的卡通表示。一个 protomer 是有色白色和其他洋红色 (残余 39-366) 和紫罗兰 (残余 367-491), 和反常的散射原子分别显示为蓝色和青色球锌和硒。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56294/56294fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>0.1 M 柠檬酸 pH 值4。0</td>
			<td>0.1 M 柠檬酸 pH 值4。5</td>
			<td>0.1 米磷酸酯 pH 值5。0</td>
			<td>0.1 米柠檬酸盐 pH 值5。5</td>
			<td>0.1 M 双三酸6</td>
			<td>0.1 M ADA pH 6。5</td>
			<td>0.1 M 拖把 pH 7</td>
			<td>0.1 M HEPES pH 7。5</td>
			<td>0.1 M 咪唑 pH 值8。0</td>
			<td>0.1 M 三 pH 8。5</td>
			<td>0.1 M 棋 pH 9</td>
			<td>0.1 M 棋 pH 9。5</td>
		</tr>
		<tr>
			<td>氯化钠 0 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
</tr></tbody></table>			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		
		<tr>
			<td>氯化钠 0.1 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
		<tr>
			<td>氯化钠 0.2 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
		<tr>
			<td>氯化钠 0.5 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
	

表1。热位移测定的缓冲成分。

Watch this Scientific Journal Video about Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092 at JoVE.com

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

一个简化的协议, 以执行广泛的生物化学和结构表征的碳水化合物基质结合蛋白从肺炎链球菌。

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

biochemical-structural-characterization-carbohydrate-transport

Nanyang Technological University

Research

JoVE Journal

Biochemistry

29.7K 次观看.  Harwell Science & Innovation Campus. 一个简化的协议, 以执行广泛的生物化学和结构表征的碳水化合物基质结合蛋白从肺炎链球菌。

视频: Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

制备及脂质立方相反应蛋白微晶交付串行飞秒晶体

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

LERLIC-MS / MS进行深入表征和谷氨酰胺的定量和天冬酰胺脱酰胺在鸟枪蛋白质组学

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

蛋白质的表征(按尺寸-排除色谱与多角度光散射相结合(SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...