作者感谢 Meytal Radzinski 为她提供了有益的讨论和对文章的批判性阅读, 以及帕特里克格里芬及其实验室成员在建立海达兴分析平台时的无限帮助。作者感谢德国-以色列基金会 (2332-1149.9/2012)、两国科学基金会 (2015056)、玛丽-居里综合赠款 (618806)、以色列科学基金会 (1765/13 和 2629/16) 和人类前沿科学计划 (CDA00064/2014) 为他们的财政支持。

1. 充分还原和全氧化蛋白的制备
<ol><li>
完全还原蛋白的制备 注: 在这里, 我们描述了一个含锌蛋白的减少, 并使用 ZnCl2溶液, 以恢复锌纳入, 降低蛋白状态。可以更换或丢弃 ZnCl2解决方案。请注意, 还原过程的时间和温度取决于蛋白质的稳定性和功能, 因此每种蛋白质都是特定的。<ol>
<li>在冰上解冻蛋白质样品, 并将其向下旋转以除去骨料。孵育样品至少1.5 小时在37°c 与5毫米和20µM ZnCl2 (多达70% 的蛋白质浓度)。 注意: 这个步骤的温度是蛋白质依赖性的, 应该调整到蛋白质的稳定性。</li>
		<li>使用除盐柱删除德勤。 注: 有不同的脱盐栏可用。下面的协议描述了使用特定脱盐柱的过程 (见材料表)。在使用其他脱盐柱之前, 我们建议检查其清除和蛋白质回收的效率, 因为一些脱盐柱可能部分吸收的蛋白质的兴趣。<ol>
<li>用磷酸钾 (KPi) 缓冲器 (40 毫米, pH 值 7.5) 平衡柱, 完全用缓冲器填满柱状物, 让缓冲液滴出。重复此过程2x。</li>
			<li>通过用镊子轻轻地将白磁盘过滤器卸下, 然后将其卸下;在用 KPi 缓冲区填充列时, 最容易删除它。用 KPi 缓冲区重新填充该列, 并将其离心 1000 x g, 3 分钟。</li>
			<li>将该列转移到一个干净的管子中, 将蛋白质样品慢慢地添加到柱中间, 并将其离心在 1000 x g 处, 2 分钟。目前, 无上无的蛋白质正在流动。</li>
		</ol>
</li>
		<li>检查其浓度 (例如, 使用紫外/可见光光谱仪), 并测量其吸光度在 280 nm。用啤酒定律计算蛋白质浓度。</li>
		<li>将一半的蛋白质样品分发到整除数中。在厌氧条件下孵化整除数 (例如, 使用厌氧室) 20 分钟, 完全清除氧气。用塑料薄膜封住管子, 将样品贮存在-20 摄氏度或-80 摄氏度, 这取决于蛋白质。 注意: 不要使用厌氧室, 管也可以用氩气闪出, 以消除氧气。</li>
	</ol>
</li>
	<li>
全氧化蛋白的制备 注: 建议从完全还原蛋白中制备氧化蛋白样品 (前面描述)。这将减少半胱氨酸氧化态的异质性。可以使用不同的氧化试剂;在这里, 我们专注于过氧化氢 (H2O2)。 注意: 避免过度氧化, 因为它可能导致不良的内二硫化物键和不可逆氧化的不同氨基酸, 包括半胱氨酸, 蛋氨酸, 酪氨酸, 和其他。<ol>
<li>到剩余的蛋白质样品, 增加5毫米 H2O2 (新鲜地稀释) 和孵化它为 3 H 在40°c, 当震动时。 注意: 这个步骤的温度是蛋白质依赖性的, 应该调整到蛋白质的稳定性。</li>
		<li>用 KPi 缓冲区 (40 mM, pH 值 7.5) 平衡列, 完全用缓冲区填充该列, 并让缓冲区滴出。重复此过程2x。</li>
		<li>通过用镊子轻轻地将白磁盘过滤器卸下, 然后将其卸下;在用 KPi 缓冲区填充列时, 最容易删除它。</li>
		<li>用 KPi 缓冲区重新填充该列, 并将其离心 1000 x g, 3 分钟。</li>
		<li>将该柱转移到一个干净的管子中, 将氧化蛋白样品慢慢地添加到柱中间, 并将其离心在 1000 x g 处2分钟;氧化蛋白现在在流动中。</li>
		<li>检查蛋白质浓度的步骤 1.2.5, 将氧化蛋白分为整除数, 并储存在-20 °c 或-80 °c, 蛋白质依赖性。</li>
	</ol>
</li>
</ol>2. 光散射聚集法
注: 本试验中的所有浓度均为陪护和基底特异, 应进行校准。所有的缓冲器应该是0.22 µm 过滤, 因为它是非常重要的, 缓冲区是没有任何粒子或气泡, 小试管是干净和无尘。使用在石英试管中放置的搅拌器是非常重要的。检查不同的搅拌器大小和形状, 以确保有效地混合整个解决方案, 而不产生不良的气泡。此外, 在实验室和设施中也有不同的 flouorospectrometers。在这里, 使用了一个特定的 fluorospectrometer (见材料表)。不同的仪器具有不同的灵敏度, 测量速度和取样参数。因此, 应该使用已知的聚集性蛋白质及其相应条件来优化精确的测量参数 (例如, 发射和激发带宽、灵敏度等)。推荐用柠檬酸合酶 (CS) 和/或荧光素素作为 nanomolar 浓度的初始基质。
<ol><li>
化学聚合试验
	<ol>
<li>在40毫米 HEPES (pH 7.5) 和4.5 米 GdnCl 中, 通过孵化12µM CS 来制备变性基质。为了保持 ph, 溶解 GdnCl 在40毫米 HEPES (pH 7.5)。</li>
		<li>打开 fluorospectrometer 软件并转到时间课程测量。将参数设置为: 温度:25 °c;λem: 360;Em 带宽: 5 毫微米;λ前: 360;前带宽: 2.5 毫微米;和数据间隔: 0.5 秒。</li>
		<li>在石英试管中加入1600µL 40 毫米 HEPES 来制备样品。将试管插入样品夹中, 并让样品达到所需的温度。</li>
		<li>设置搅拌到 600 rpm, 并开始测量, 直到建立基线。为整个测量保持搅拌。</li>
		<li>要测量 cs 聚合在没有伴侣的情况下, 在120s 进入测量, 添加 (轻轻, 但迅速) 10 µL 变性 CS (最终浓度为 75 nM)。继续测量1200秒。</li>
		<li>要测量 CS 聚合在存在 Hsp33, 在六十年代入测量, 增加 Hsp33 (最后的集中300毫微米)。在另外的六十年代以后, 增加10µL 变性 CS (最后的集中75毫微米)。继续测量1200秒。 注: 添加衬底或陪护时, 为了避免气泡的插入, 请使用10µL 吸管。</li>
	</ol>
</li>
	<li>
热聚合试验 注: 热聚集试验的温度取决于蛋白质的稳定性, 并应独立调整到每种蛋白质。<ol>
<li>打开 fluorospectrometer 软件并转到时间课程测量。按如下方式设置参数: 温度:43 °c;λem: 360;发射带宽: 5 nm;λ前: 360;励磁带宽: 2.5 毫微米;和数据间隔: 0.5 秒。</li>
		<li>在石英试管中加入1600µL 预热40毫米 HEPES, 制备样品。将试管插入样品夹中, 并让样品达到43摄氏度。</li>
		<li>设置搅拌到 600 rpm, 并开始测量, 直到建立基线。为整个测量保持搅拌。</li>
		<li>要测量 cs 聚集在没有陪护, 在120s 入测量, 轻轻地增加 cs (最后集中125毫微米) 和继续测量 1200 s。</li>
		<li>要测量 CS 聚合在存在 Hsp33, 在六十年代入测量, 增加 Hsp33 (最后的集中600毫微米)。在另外的六十年代以后, 增加 CS (最后集中125毫微米)。继续测量1200秒。 注: 添加衬底或陪护时, 避免气泡的插入。</li>
	</ol>
</li>
	<li>
Kfits 的数据分析与噪声去除 注: Kfits 在使用Kfits之前已被描述在 Rimon等。45<ol>
<li>上载数据文件。 注: 输入是以文本逗号分隔或制表符分隔格式的光散射测量产生的原始结果。</li>
		<li>要删除任何噪音, 请选择 "分析参数"。使用自动最佳模型(推荐), 标记噪音始终高于信号标志。</li>
		<li>使用绿色和红色可调整线手动删除明显的异常点;这一步将过滤在绿线和红线下面的噪音。</li>
		<li>设置基线和拟合曲线。应用噪声阈值后, 下载已处理的数据。</li>
	</ol>
</li>
</ol>3. 氢-氘交换质谱法
<ol><li>缓冲液和蛋白质样品的制备 (Hsp33 和 Hsp33-CS 络合物)
	<ol>
<li>将蛋白质样品稀释到1毫克/毫升的最终浓度, 在25毫米的三盐酸缓冲液中 pH 7.5, 并将其转移到1.5 毫升的瓶子。</li>
		<li>以 1:1. 5 为43摄氏度, 用 CS 孵化 Hsp33 制备蛋白质基质复合样品。<ol>
<li>以步骤明智的方式添加 CS, 以避免任何快速聚合, 并确保 Hsp33 和热展开 CS 之间有一个卓有成效的绑定。</li>
			<li>使用至少四个步骤 (即每次添加一个季度的最终卷), 并孵化的样本, 每增加15分钟后, 允许 CS 保护由 Hsp33。</li>
		</ol>
</li>
		<li>用30分钟离心法去除任何骨料, 在 1.6万 x g 处4摄氏度。 注: 新的蛋白-基质配合物必须准备新鲜。衬底应逐渐增加;否则, 将发生聚合。温度、基底和基质浓度是特定于蛋白质的。</li>
		<li>准备一个缓冲 H (25 毫米三 HCl, pH 值 7.5), 作为氘化控制, 和缓冲 D (25 毫米三 DCl, pH 7.09), 这是氘化缓冲区。还准备一个新的淬火缓冲器 (150 毫米 TCEP, 3 米 GdnCl, 0.1% 甲酸)。 注: 在胃蛋白酶消化后, 为了获得其最大的序列覆盖率, 应对感兴趣的蛋白质进行最佳的淬火缓冲。</li>
		<li>把所有的缓冲器和样品转移到瓶子里, 放在适当的盘子里。保持缓冲器 H 和 D 在25°c (托盘25°c);另一方面, 保持样品和淬火缓冲器在 0-2 °c (托盘0°c)。将样品放在150µL 玻璃插入物中 (见材料表), 然后将其转移到小瓶中。</li>
	</ol></li>
	<li>仪器的准备 注: 质谱仪附带两个附带的软件程序: 一个控制水泵, 另一个控制质谱仪 (参考材料表)。接下来的步骤将使用这两个软件程序来描述。<ol>
<li>手动打开所有冷却设备。一旦所有冷却系统达到其目标温度, 手动切换的高性能液相色谱 (HPLC) 和加载泵。 注: 在我们的例子中, 这些包括两个冷却浴缸和一个冷却箱 (其中包含陷阱和分析列)。一个冷却装置冷却托盘在0°c, 而另一个保持托盘在25°c;冷却箱的温度为 1-2 摄氏度。</li>
		<li>打开控制水泵的软件, 以及控制质谱仪的软件;请确保 MS 处于待机状态。</li>
		<li>从 MS 源断开 HPLC 出口阀, 首先用 "三清洁剂" 溶液 (1% 甲酸, 33% 乙腈, 33% 异丙醇, 33% 甲醇) 清洗系统, 然后用缓冲 B (80% 乙腈, 0.1% 甲酸), 最后用缓冲器 a (0.1%甲酸, pH 值 2.25), 然后将胃蛋白酶柱插入系统中。如果更改缓冲区, 请确保在继续之前清除两个泵。</li>
		<li>确保两种泵的流速为0.1 毫升/分, 压力稳定。</li>
		<li>用稳定的流量和稳定的压力清洗系统后, 将高效液相色谱插座插入 ms 源并打开 ms。</li>
	</ol></li>
	<li>质谱仪参数
	<ol>
<li>将肽电离设置为电喷雾电离 (ESI) 在175°c, 鞘气体流动在 17, 辅助气体发光在 2, 并且喷雾电压在4.5 伏 (补充图 1)。</li>
		<li>为非氘示例设置参数, 如下所示。<ol>
<li>设置参数: 扫描范围 m/z 到 300-1500;决议到 7万;自动增益控制目标 (AGC) 到 106;和最大注射时间 (它) 在100毫秒。 注: 5 最强的离子与充电状态在 +2 和 +6 之间 (他们的强度高于 1.3 x 104) 和一个动态窗口30在将被隔绝。</li>
			<li>在具有归一化碰撞能量 (HCD) 的多碰撞细胞中, 用高能碰撞离解 () 进行离子碎片化, 等于28。通过串联 MS 检测片段离子的分辨率为 3.5万, AGC 目标为 105, 最大值为60毫秒, 隔离窗口为2.0 米/z。</li>
		</ol>
</li>
		<li>对于氘样本, 仅使用 MS1的分辨率较高的14万和其他类似的参数。</li>
	</ol></li>
	<li>运行实验
	<ol>
<li>以下列方式设置托盘。<ol>
<li>将缓冲 H 和 D 放入托盘25°c, 然后将样品和淬火缓冲器放在0°c 托盘中。</li>
			<li>放置 x 空瓶, 对齐在两个托盘, 其中 x = 样本数 x 的时间点数。 注: 在25°c 托盘, 空瓶作为反应瓶, 并在托盘0°c, 他们作为淬火瓶。</li>
			<li>每个空瓶子都包含一个空的玻璃插入物;在实验之间丢弃和替换所说的插入。 注意: 为了以最有效和最耗时的方式运行海达兴实验, 使用了一个允许同时运行示例的软件 (辅助图 2)。接下来的步骤将使用此软件进行描述。</li>
		</ol>
</li>
		<li>打开软件。设置程序参数以执行以下操作。<ol>
<li>根据所选的时间点 (如1、3、18、40和100分钟) 在25摄氏度上孵化每个样品 (5 µL) 和45µL 的缓冲 D。用45µL 的缓冲 H 来孵化非氘样品。</li>
			<li>孵化后立即将50µL 的氘蛋白混合成50µL 的冰冷淬火缓冲器, 并将其注入固定的胃蛋白酶柱 (长度为20毫米, 直径2.0 毫米)。</li>
			<li>洗脱从胃蛋白酶柱到前柱的任何肽, 洗涤它与缓冲 a 为6分钟以50µL/分钟的速度. 在冷却箱中保持缓冲 a 0 摄氏度。</li>
			<li>洗脱的肽从前柱到 C18 分析柱 (C18 柱, 130 Å, 1.7 µm, 2.1 毫米 x 50 毫米, 保持在0°c) 和分开他们通过应用一个线性梯度乙腈在100µL/分钟. 用乙腈100% 作为缓冲 B 运行乙腈梯度: 7 分钟在 2%, 7 分钟在 10-30%, 2.5 分钟在 30-90%, 1.5 分钟在 90%, 快速梯度为1分钟在 90-8%, 并且最后平衡列为4分钟在2%。</li>
		</ol>
</li>
		<li>生成一个运行序列 (请参见辅助图 2): 在托盘中输入所有的时间点、示例名称和位置, 以及缓冲区名称和位置。 注意: 软件允许将所有不同的运行时间对齐到一个程序中, 一次运行多个示例, 从而有效地减少运行时间。</li>
	</ol></li>
	<li>数据分析 注意: 在数据分析过程中, 我们使用了几个软件程序, 包括蛋白质组发现者和MaxQuant (自由软件), 用于对序列覆盖进行多肽识别和分析, 以及海达兴工作台,自由软件, 允许分析的氘纳入蛋白质, 并比较海达兴率之间的不同组的实验。接下来的步骤将使用这些软件程序进行描述。<ol>
<li>通过软件运行非氘控制样本来分析样品的肽覆盖率 (补充图 3)。使用以下参数设置软件。<ol>
<li>使用 Sequest HT 法与无酶 (不特异) 劈裂。寻找 4-144 氨基酸的肽, 其质量耐受性为前体的离子和片段的 7 ppm 和 0.5 Da。允许动态的蛋氨酸氧化。</li>
			<li>创建一个蛋白质的数据库, 通过该软件可以确定肽覆盖率。以文本格式导出已识别的多肽。</li>
		</ol>
</li>
		<li>使用海达兴工作台免费软件58分析氘结果。<ol>
<li>通过插入蛋白质序列和肽覆盖率文件, 打开蛋白质编辑器并定义蛋白质。</li>
			<li>接下来, 打开安装实验向导编辑器并输入所请求的所有输入。 注意: 该程序需要样本数、时间点数、复制次数、缓冲区的 pH 值和温度。</li>
			<li>最后, 输入有关质谱仪使用的参数, 然后程序生成检测到的多肽的列表。 注意: 该软件检测到 "海达兴" 肽, 检查同位素簇, 并证明了氘化在样品中的清晰可视化。</li>
		</ol>
</li>
	</ol></li>
	<li>数据演示
	<ol>
<li>使用PyMol软件或任何其他可视化软件, 对结构进行恭敬氘化吸收的区域进行标记。</li>
		<li>介绍氘吸收水平, 而不是 B 因子值。 注: 基本教程可以在 https://pymolwiki.org/index.php/Practical_Pymol_for_Beginners 中找到。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

在本文中, 我们提供了分析氧化还原依赖性伴侣活动的方法, 以及对蛋白质结合后结构变化的描述。这些是补充的方法, 以确定潜在的伴侣-基质化合物和分析潜在的互动网站。
在这里, 我们应用这些协议来描述一个复杂的氧化还原调节伴侣 Hsp33 与一个良好研究的陪护基质 CS。我们提出了两种不同的蛋白质聚集分析, 它们的动力学和初始状态不同: 在化学变性过程中, 基体从变性?...

细胞经常遇到活性氧的积累 (ROS) 产生作为呼吸的副产品1,2, 蛋白质和脂质氧化3,4和其他过程5, 6,7。尽管 ros 在多种生物过程中起着有益的作用, 如细胞信号8、9和免疫应答10, ros 生产与解毒之间的不平衡可能会发生, 导致氧化压力7。活性氧的生物学指标是蛋白质、脂质和核酸, 其氧化作用影响其结构和功能。因此, 细胞氧化剂的积累与多种病症有密切的联系, 包括癌症9,11, 炎症12,13, 衰老14,15, 并已发现参与神经退行性疾病的发病和进展, 如阿尔茨海默氏症, 帕金森氏症, ALS 疾病16,17,18。
新合成的和成熟的蛋白质对氧化反应高度敏感, 因为它们的侧链有可能有害的修饰, 它的形状蛋白质结构和功能19,20。因此, 氧化应激通常导致广泛的蛋白质失活, 错误折叠和聚集, 最终导致细胞死亡。一个优雅的细胞策略, 以应付潜在的损害蛋白质氧化是利用氧化还原依赖的伴侣, 这抑制了广泛的蛋白质聚集, 而不是形成稳定的配合与错误折叠客户端蛋白21 ,22,23。这些一线防御伴侣是迅速激活的现场特定氧化 (通常是半胱氨酸残留), 转化成有效的抗聚集分子24。由于氧化应激导致呼吸抑制和细胞 atp 水平下降25, 典型的 atp 依赖伴侣在氧化应激条件下的有效性较差25,26 ,27。因此, 氧化还原活化的 ATP 独立伴侣在维持蛋白质稳态的同时, 在细菌和真核生物中的氧化剂积累 (例如, 细菌中的 Hsp3328和佛罗里达29 , Get3 30) 中起着至关重要的作用.在酵母, peroxiredoxins31在真核生物)。这些伴侣的活动很大程度上取决于由特定地点的氧化引起的可逆结构构象变化, 它揭示了涉及识别错误折叠客户蛋白的疏水性区域。
基于伴侣-基质相互作用32、33的动态和异性质, 研究抗聚集机制和控制伴侣对客户蛋白质识别的原则并不容易, 34,35,36,37。然而, 受压力调节的伴侣有机会提高我们对抗聚集功能的理解, 因为他们的能力: 1) 获得两种不同形式的伴侣, 活跃 (如, 氧化) 和非活动 (例如,减少), 与引入或消除的压力条件容易切换 (例如, 氧化剂和还原剂), 2) 有广泛的基质, 3) 形成高度稳定的复合体与客户的蛋白质, 可以评估不同的结构方法, 和 4) 只专注于基板的识别和释放, 介导的氧化还原依赖性构象变化, 因为大多数这些伴侣缺乏折叠能力。
在这里, 我们分析的细菌氧化还原调节陪护 Hsp33's 抗聚集活性, 一个重要组成部分的细菌防御系统抗氧化诱导蛋白聚集28。减少时, Hsp33 是一种紧密折叠的锌结合蛋白, 无陪护活动;然而, 当暴露在氧化应激, Hsp33 经历了广泛的构象变化, 暴露其基质结合区38,39。氧化后, 与 C 末端域四高度保守的半胱氨酸残留物密切相关的锌离子被释放40。这就导致了两个二硫化物键的形成, C 端域的展开, 以及相邻链接器区域41的不稳定。C 终端和链接器区域高度灵活, 被定义为内在的或部分无序的。当返回到非压力条件, 半胱氨酸减少, 陪护返回其本机折叠状态, 没有反聚集活动。伴侣的复性导致了束缚的顾客蛋白质的进一步展开和不稳定, 触发它的转移到规范化的伴侣系统, DnaK/J, 为复性38。对 Hsp33's 相互作用站点的分析表明, Hsp33 使用其带电的无序区域以及链接器和 N 终端域上的疏水区域来捕获错误折叠的客户端蛋白质并防止其聚合38,42. 在折叠状态下, 这些区域由折叠链接器和 C 终端域隐藏。有趣的是, 链接器区域充当 Hsp33's 折叠和非活动状态的看门人, "感知" 其相邻的 C 终端域34的折叠状态。一旦突变 (无论是点突变或全序列摄动), Hsp33 被转化为组成性主动陪护者, 无论其氧化还原敏感性半胱氨酸43的氧化还原状态。
这里提出的协议允许监测 Hsp33's 氧化还原依赖的陪护活动, 以及映射构象变化的激活和结合的客户端蛋白。这种方法可以适应研究其他伴侣-客户识别模型以及非伴侣蛋白-蛋白质相互作用。此外, 我们提出了制备完全还原和氧化的伴侣的协议, 可用于其他氧化还原开关蛋白的研究, 以揭示蛋白质氧化对蛋白质活性的潜在作用。
具体来说, 我们描述了一个程序, 以监测伴侣活动的体外, 并确定其基底特异性在不同类型的蛋白质聚集 (化学或热诱导) 使用光散射 (LS) 测量的fluorospectrometer44。在聚合过程中, 由于浊度的增加, 360 nm 的光散射迅速增加。因此, 可以在这种波长上以时间依赖性的方式监视聚合。LS 是一种快速、灵敏的检测蛋白质聚集的方法, 因而利用 nanomolar 浓度对感兴趣的蛋白质进行抗聚集活性, 使蛋白质聚集相关的动力学参数在不同的条件。此外, 此处描述的 LS 协议不需要昂贵的仪器, 并且可以很容易地在任何实验室中建立。
然而, 获得 "清洁" 的动力学曲线, 并从这样的光散射实验中提取蛋白质的动力学参数是相当有挑战性的, 因为噪声和气泡和大骨料产生的大量异常点。为了克服这一障碍, 我们提出了一种新的图形工具, Kfits45, 用于降低不同的动力学测量的噪音水平, 特别适合蛋白质聚集动力学数据。该软件为早期评估结果提供了初步的动力学参数, 允许用户在不影响其动力学特性的情况下快速 "清除" 大量数据。Kfits是在 Python 中实现的, 在45的开源中可用。
这一领域的一个具有挑战性的问题是, 在伴侣和他们的客户蛋白质之间绘制相互作用的站点, 了解伴侣如何识别广泛的错误折叠基质。当研究高度动态的蛋白质复合物时, 这个问题会更加复杂, 这涉及到内在紊乱的伴侣和容易聚集的基质。幸运的是, 结构质谱在过去十年中已有了显著的进步, 并成功地提供了有用的方法和工具来分析蛋白质识别46中所涉及的结构可塑性和地图残留,47,48,49. 在这里, 我们提出一个这样的技术-氢-氘交换质谱 (海达兴)-这使得在结构构象上的残留水平的变化映射到蛋白质修饰或蛋白质/配体结合35, 50,51,52,53,54,55。海达兴-MS 使用氘的连续交换骨干氢, 其速率受化学环境、可达性和共价键和非共价键56的影响。海达兴-MS 跟踪这些交换过程使用的氘溶剂, 通常是重水 (D2O), 并允许测量的基础上分子量的变化后, 氢的氘交换。氢氘交换速率的减慢可能是氢参与氢键或简单地从空间障碍中产生的, 这表明结构57的局部变化。在配体结合或平移后修改后的变化也可能导致氢环境的差异, 具有约束力, 导致氢氘交换 (海达兴) 率的差异46,53。
我们将这项技术应用到 1) 地图 Hsp33 地区迅速展开后, 氧化, 导致活化 Hsp33, 和 2) 定义的潜在结合界面 Hsp33 与其全长错误折叠基质, 柠檬酸合酶 (CS)38。
本手稿所描述的方法可用于研究蛋白质在体外的氧化还原依赖性功能, 定义抗聚集活性和结构变化 (如有) 在蛋白质功能中的作用。这些方法可以很容易地适应不同的生物系统, 并应用于实验室。

<table><tbody><tr><td>&lt;strong&gt;Chemicals, Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile HPLC plus</td><td>Sigma Aldrich</td><td>34998-2.5L</td><td>solvent</td></tr><tr><td>Formic acid Optima LC/MS</td><td>Fisher Chemicals</td><td>A117-50</td><td>solvent supplement</td></tr><tr><td>Isopropyl alcohol, HPLC grade</td><td>Fisher Chemicals</td><td>P750717</td><td>solvent</td></tr><tr><td>Methanol</td><td>Fisher Chemicals</td><td>A456-212</td><td>solvent</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma Aldrich</td><td>252859</td><td>buffer</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma Aldrich</td><td>76-05-1</td><td>solvent</td></tr><tr><td>Water for HPLC</td><td>Sigma Aldrich</td><td>270733-2.5L-M</td><td>solvent</td></tr><tr><td>ZnCl2, Zinc Chloride</td><td>Merck</td><td>B0755416 308</td><td>reagent</td></tr><tr><td>DTT</td><td>goldbio</td><td>27565-41-9</td><td>reducing agent</td></tr><tr><td>PD mini trap G-25 columns GE healthcare</td><td>GE healthcare</td><td>29-9180-07</td><td>desalting column</td></tr><tr><td>Potassium Phosphate</td><td>United states Biochemical Corporation</td><td>20274</td><td>buffer</td></tr><tr><td>Hydrogen peroxide 30%</td><td>Merck</td><td>K46809910526</td><td>oxidizing agent</td></tr><tr><td>citrate synthase</td><td>sigma aldrich</td><td>C3260</td><td>substrate</td></tr><tr><td>HEPES acid free</td><td>sigma aldrich</td><td>7365-45-9</td><td>buffer</td></tr><tr><td>Gndcl</td><td>sigma aldrich</td><td>G3272-500G</td><td>denaturant</td></tr><tr><td>Deuterium Chloride Solution</td><td>sigma aldrich</td><td>543047-10G</td><td>buffer</td></tr><tr><td>Deuterium Oxide 99%</td><td>sigma aldrich</td><td>151882-100G</td><td>solvent</td></tr><tr><td>TCEP</td><td>bioworld</td><td>42000058-2</td><td>reducing agent</td></tr><tr><td>150uL Micro-Insert with Mandrel Interior &amp;amp; Polymer Feet, 29*5mm</td><td>La-Pha-Pack -Thermo Fischer Scientific</td><td></td><td></td></tr><tr><td>1.5mL Clear Short Thread Vial 9mm Thread, 11.6*32mm</td><td>La-Pha-Pack -Thermo Fischer Scientific</td><td></td><td></td></tr><tr><td>quartz cuvette</td><td>Hellma 101-QS</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Jasco FP-8500 Fluorospectrometer</td><td>Jasco</td><td></td><td></td></tr><tr><td>Thermomixer Comfort</td><td>Eppendorf</td><td>13058/0</td><td></td></tr><tr><td>Heraeus Megafuge 16R, bench topCentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>pH meter , PB-11 sartorius</td><td>Sartorius</td><td>13119/0</td><td></td></tr><tr><td>AffiPro Immobilized Pepsin column (20mm length, 2.0mm diameter).</td><td>AffiPro</td><td></td><td></td></tr><tr><td>Waters Pre-column (ACQUITY UPLC BEH C18 VanGuard 130 &amp;Aring;, 1.7um, 2.1mmx5mm)</td><td>Waters</td><td></td><td></td></tr><tr><td>C18 analytical column (ACQUITY UPLC Peptide BEH c18 Column, 130 &amp;Aring;, 1.7um, 2.1mmx50mm)</td><td></td><td></td><td></td></tr><tr><td>Vinyl Anaerobic chamber with Airlock door</td><td>COY</td><td></td><td></td></tr><tr><td>Q-exactive-orbitrap mass spectrometer</td><td>Thermo-Fischer Scientific</td><td></td><td></td></tr><tr><td>PAL system LHX - robotic system for handling HDX samples</td><td>PAL system</td><td>https://www.palsystem.com/index.php?id=840</td><td></td></tr><tr><td>Dionex Ultimate 3000, XRS pump</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dionex AXP-MS auxiliary pump</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software, Software Tools, Database search&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kfits: Fit aggregation Data</td><td>http://kfits.reichmannlab.com/fitter/</td><td></td><td></td></tr><tr><td>Thermo Scientific Xcalibur software</td><td>https://www.thermofisher.com/order/catalog/product/OPTON-30487</td><td></td><td></td></tr><tr><td>Q Exactive MS Series Tune Interface (Tune)</td><td>https://tools.thermofisher.com/content/sfs/brochures/WS-MS-Q-Exactive-Calibration-Maintenance-iQuan2016-EN.pdf</td><td></td><td></td></tr><tr><td>Chronos software (Axel Semrau)</td><td>http://www.axel-semrau.de/en/Software/Software+Solutions/Chronos-p-966.html</td><td></td><td></td></tr><tr><td>Proteome Discoverer V1.4 software</td><td>https://www.thermofisher.com/order/catalog/product/OPTON-30795</td><td></td><td></td></tr><tr><td>HDX workbench software</td><td>http://hdx.florida.scripps.edu/hdx_workbench/Home.html</td><td></td><td></td></tr></tbody></table>

defining hsp33's redox-regulated chaperone activity and mapping conformational changes on hsp33 using hydrogen-deuterium exchange mass spectrometry

生物体经常需要在其生命周期中应对波动的环境, 包括温度、pH 值的变化、活性氧的积累等等。这些波动可能导致广泛的蛋白质展开, 聚集和细胞死亡。因此, 细胞已经进化出一个动态和应力特异的分子伴侣网络, 在压力条件下维持一个 "健康的" 蛋白质组。ATP 独立伴侣构成了一个主要的类分子伴侣, 作为一线防御分子, 防止蛋白质聚集的压力依赖性的方式。这些伴侣共有的一个特点是, 他们有能力利用结构可塑性来错误折叠客户的压力特定的激活、识别和释放。
本文重点讨论了一种这种内在紊乱的伴侣的功能和结构分析, 即细菌氧化还原调节 Hsp33, 它在氧化应激过程中保护蛋白质不聚集。在这里, 我们提出了一个不同的技术工具箱, 研究氧化还原调节的陪护活动, 以及映射的构象变化的监护人, 其活动的基础。具体来说, 我们描述了一个工作流, 包括制备完全还原和完全氧化的蛋白质, 然后分析的伴侣抗聚集活动的体外利用光散射, 集中在程度抗聚集活性及其动力学。为了克服聚集分析过程中累积的异常现象, 我们描述了Kfits的用法, 这是一种新的图形化工具, 可以方便地处理动态测量。该工具可以很容易地应用于其他类型的动力学测量, 以消除异常和拟合动力学参数。为了使功能与蛋白质结构相关联, 我们描述了一种结构质谱技术、氢-氘交换质谱法的建立和工作流程, 该方法允许对监护人的构象变化进行映射, 并基质在 Hsp33 活动的不同阶段。同样的方法也适用于其他蛋白质和蛋白质配体的相互作用。

所提出的两种方法使人们有可能遵循伴侣与其基质之间蛋白质相互作用的动力学活动和动力学。此外, 还原氧化协议允许准备一个完全还原和完全氧化的伴侣, 使更深入地了解氧化还原依赖紊乱的伴侣的活化机制。
首先, 我们使用光散射, 以检查与氧化还原相关的活动的陪护。光散射是在化学和热变性 CS 蛋白存在氧化 (活跃) 或?...

Watch this Scientific Journal Video about Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry at JoVE.co

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

生物体在其一生中遇到的最具挑战性的压力条件之一是氧化剂的积累。在氧化应激过程中, 细胞大量依赖分子伴侣。在这里, 我们介绍了用于研究氧化还原调节的抗聚集活性的方法, 以及通过海达兴-MS 来监测调节陪护功能的结构变化。

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

defining-hsp33-s-redox-regulated-chaperone-activity-mapping

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8.6K 次观看.  The Hebrew University of Jerusalem. 生物体在其一生中遇到的最具挑战性的压力条件之一是氧化剂的积累。在氧化应激过程中, 细胞大量依赖分子伴侣。在这里, 我们介绍了用于研究氧化还原调节的抗聚集活性的方法, 以及通过海达兴-MS 来监测调节陪护功能的结构变化。

视频: Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

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生物化学

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

毫秒氢/氘交换质谱法用于生理条件下α-突触核蛋白结构动力学的研究

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...