本文所描述的 X 射线衍射实验是使用同步辐射08号和 08-BM 在加拿大光源上进行的, 该试验由加拿大创新基金会、加拿大自然科学和工程研究委员会支持,萨斯喀彻温省大学、萨斯喀彻温省政府、加拿大西部经济多样化、加拿大国家研究委员会和加拿大卫生研究所。我们要感谢为患病儿童医院提供的结构 & 生物物理核心设施, 以便进入国贸中心和 BLI 仪器。J.E.O. 得到了加拿大卫生研究院班廷博士后奖学金 BPF-144483 的支持。Vanier 是加拿大大学研究生奖学金获得者, 加拿大卫生研究院的一项研究生奖学金。这项工作得到了加拿大卫生研究院的 PJT-148811 (J.-p.j) 的资助。这项研究的一部分原因是加拿大研究椅项目提供的资金(J.-p.j)。

作者声明没有相互竞争的利益。

膜定位糖蛋白是细胞功能的关键, 是有吸引力的治疗目标。在这里, 我们提出了一项关于膜糖蛋白的早期发育的结构和生物物理特性的协议, 这两种方法单独和复杂的小分子配体和晶圆碎片。我们已经成功地使用这个协议来确定人 CD2228的细胞外部分的三 N 末端最透明的区域的晶体结构, 一个关键的联合受体在 B 细胞参与保持体液免疫在检查79。我们还描绘了 CD22 与天然配体α2-6 唾液乳糖的结合部位, 并定义了一种治疗抗体对人类 CD22 的识别模式。这些结果提供了对 Siglecs 家族的一个关键成员的结构-功能关系的洞察力, 它限制了 B 细胞的表达, 并为发展新的 CD22 靶向小分子和抗体基础的分子路线图疗法。虽然该协议成功地用于含有 B 细胞受体, 我们建议, 我们的方法可以适用于结构和生物物理特性的任何膜糖蛋白与一个独特的领域组织。在这种情况下, 构造设计和组合 N 链接糖突变 (无论是 Gln 或亚拉巴马州) 可以评估, 以找到一个适合晶体生长和高分辨率衍射的结构。
获得一个均匀和纯糖蛋白样品是至关重要的晶体生长和 x 射线衍射, 以及下游生物物理特性。n-链多糖存在于糖蛋白本身是异构的, 并可能导致构象和化学异质性内的糖蛋白, 可以阻止晶体形成。为了减少这种微异质性, 引入点突变的策略, 以去除预测将 n-链多糖的 Asn 残留物, 或者使用突变细胞线 (如 HEK293S), 然后用 endoglycosidases (如 EndoH) 进行治疗, 可以大大改善结晶成功15,21,22。在本议定书中, 我们讨论了可溶性糖蛋白和晶圆的纯化, 分泌到细胞中清。糖蛋白分泌提供了一个相对简单的途径, 以纯度, 不需要细胞裂解或添加苛刻的化学品或洗涤剂。在细胞收获之后获得的细胞上清, 然后直接运行在一个对感兴趣的蛋白质有亲和力的专栏 (例如,镍 NTA 为他标记的糖蛋白, 或 LC 亲和为晶圆碎片)。然而, 根据使用的专栏和细胞上清的条件 (例如, pH 值), 对柱感兴趣的蛋白质的结合能力可能受到影响。如果是这种情况, 可能需要集中和缓冲交换细胞上清, 以改善绑定到该列。此外, 强烈建议使用净化过程中的质量控制步骤, 以帮助评估蛋白质纯度。运行一个 SDS 页凝胶或所有样品的西部污点 (在纯化步骤之前、过程中和之后), 可以对建议的纯化方案是否适合感兴趣的蛋白质产生洞察力。如果在 SDS 页上可见污染带, 或者在提纯过程中获得了几个物种 (例如,在大小排除上有几个峰值), 则应考虑额外的净化步骤,例如离子交换色谱, 以获得在纯净和增加机会下游结晶80。
对于大分子结晶, 通常关键是要获得高产量的蛋白质的利益, 以便筛选大量的潜在的结晶条件, 在高蛋白质浓度, 以找到合适的晶体命中。一般来说, 这里讨论的 HEK293 细胞线 (HEK293F 和 HEK293S) 是健壮的表达系统, 可以很容易地放大, 以产生更多的样本, 必要的。然而, 有可能的蛋白质的兴趣可能没有足够的表达在这些细胞系。在这些情况下, 其他细胞系, 如 Expi293 细胞81,82, 已被发现显示高水平的蛋白质表达, 应该被认为是一种替代品。
如果有良好的顺序, 衍射晶体是没有得到以下几个结构的兴趣, 尽管高纯度的蛋白质, 可能需要扩大结晶技术, 以促进晶体形成。结果表明, nanobodies 的抗体和微晶片段可以是极好的结晶增强剂, 并促进有序的晶体填料83,84,85。这些片段可以表达和纯化为同质性, 并用于一个复杂的与感兴趣的蛋白质促进结晶。重要的是, 如10节所述产生的晶圆碎片可能有形成非功能 LC 脂肪酸86的倾向。这些脂肪酸是污染物, 应在净化过程中去除。根据我们的经验, LC 脂肪酸通常有一个不同的保留量的大小排除, 或洗脱作为一个独特的峰值离子交换色谱, 从而可以从晶圆净化-但这并不总是如此。如果这些技术不足以去除脂肪酸从晶圆纯化, 额外的纯化方法, 如蛋白质 G 亲和纯化, 可用于提高纯度。
另一种方法是与晶圆碎片进行相互配合, 记录良好的技术 (如随机矩阵 microseeding) 可以提高获得有序晶体63、70的几率。该方法包括将少量粉碎的, 次优晶体加入结晶条件中, 提供晶体的核化以促进晶体的生长。这可以使用感兴趣的蛋白质晶体, 或具有类似的领域体系结构和三重结构。此外, 随机矩阵 microseeding 可以在试图单独结晶的蛋白质, 或复杂的一个晶圆或小分子的利益。最近的研究进展, 在低温电子显微镜也使这项技术成为一个有吸引力的替代 X 射线晶体学获得高分辨率结构信息的分子具有适当的特征87,88, 89,90,91。
当 X 射线衍射数据集的分阶段被 MR 所失败时, 可能需要 HA 浸泡以通过异常色散或同构替换来解决相位问题。检测蛋白质的氨基酸序列可以为 HA 衍生的策略提供线索, 包括最佳的结合 pH 值。特别是, 蛋白质内配对的半胱氨酸可以专门约束含有汞的 HA 化合物。用 ha 化合物浸泡本族晶体是一个迭代过程, 用于确定最佳 HA 化合物的特性、浓度和所需的孵化时间。如果初始浸泡尝试不产生良好的衍射晶体, 含有 ha 适合分阶段, 可能需要引入氨基酸替代, 以提高 ha 结合的可能性, 改善异常信号。例如, 含有游离半胱氨酸残留物的突变可以有效地结合汞、Au、Pt 或 Pb. 蛋白质在硒-蛋氨酸补充培养基中的异常分期的表达广泛用于异常分期, 然而硒-蛋氨酸的等效系统在悬浮92、93的哺乳动物细胞中不易获得, 是未来发展的一个领域。
一旦获得感兴趣的糖蛋白的 unliganded 结构, 就可以用小分子配体浸泡晶体来获得免疫受体-配合物的结构。这些数据为合理设计更特异和高亲和性配体提供了一个蓝图, 可以作为小分子治疗, 并提供高分辨率洞察到糖蛋白的生物学功能。当试图浸泡糖蛋白晶体与小分子配体的兴趣, 检查的 unliganded 晶体结构可以表明是否浸泡应该是可能的。如果在配体结合部位周围或周围区域发现接近的晶体填料接触, 则在配体结合时会发生构象变化, 浸泡可能会有问题。在这种情况下, 应执行其他方法, 如共结晶的蛋白质配体复合物。

表面蛋白在细胞功能中起重要作用。通过它们的胞外域, 这些膜蛋白可以调节细胞的相互作用, 黏附力, 传输和信号1,2。这些蛋白质的胞外定位使它们成为治疗多种疾病的疗法的诱人靶点, 包括癌症和自身免疫性疾病3,4,5,6,7. 人类膜蛋白 ectodomains 最常见的褶皱之一是免疫球蛋白样 (Ig) 褶皱, 由七或更多的β链组成, 排列成两个β片8,9。一般情况下, 含膜糖蛋白是多域结构, 在细胞膜蛋白10的胞外部分按顺序排列。这些细胞表面蛋白, 特别是 n-和 O 连锁糖基化的转化后的修饰, 在它们的调节、折叠、分泌和功能11中发挥了重要作用。为了提高我们对其功能的了解, 更好地设计能够针对它们的治疗方法, 需要技术, 使其具有详细的分子特征。在这里, 我们提出了技术的组合, 允许生物物理 (biolayer 干涉 (BLI) 和等温滴定量热 (贸易中心)) 和结构 (X 射线晶体学) 的细胞外域的表征膜糖蛋白, 单独和在复杂与他们的生物相关的配体和治疗分子 (图 1)。
n-链接糖基化是哺乳动物蛋白质的最常见的后翻译修改, 并发生在蛋白质成熟期间内质网和高尔基12,13。细胞系, 如人胚肾 (HEK) 293 细胞, 已开发用于重组表达大量糖化哺乳动物蛋白14,15。这一细胞系是以悬浮的形式发展的, 它允许将蛋白质的生产与粘附细胞线相比更容易地扩大到更大的数量。在这里, 我们利用两个 HEK293 细胞系: HEK293F 和 HEK293 Gnt I/- (HEK293S), 这是不同的 n-acetylglucosaminyl 转移酶 i (Gnt) 在后者。反过来, 复杂多糖的生产 (如 HEK293F 中所见) 是不可能的, 而是高甘露糖型多糖 (主要是人5GlcNAc2) 居住在 N 链接糖站点18,19,20.同时使用这两个细胞线可以研究糖大小和复杂性对生物学功能和治疗靶向的影响。事实上, HEK293F 细胞产生的糖蛋白将有更大, 更复杂的多糖相比, HEK293S 细胞生成的糖蛋白。HEK293S 细胞产生的糖蛋白更易于结晶, 因为它们的 n-链多糖的化学和构象异质性降低。为了进一步改善结晶性能, HEK293S (而不是 HEK293F) 细胞中产生的糖蛋白可以用酶 endoglycosidase h (内 h) 处理, 从而导致高甘露糖多糖的分裂, 只有一个 n-葡糖 (GlcNAc)基团仍然在每 N 链接的糖基化站点21,22。其他方法也可用于限制细胞内的 n-糖处理, 例如在糖蛋白表达过程中添加 glycosyltransferase 抑制剂, 包括 kifunensine23。替代方法包括在 HEK293F 细胞中表达原生糖蛋白 (deglycosylation), 然后使用肽 n-糖苷酶 F (PNGaseF) 的酶。然而, deglycosylation 与 PNGaseF 已被证明是不太有效的当地条件下, 增加聚集在一些蛋白质;在治疗后, 当蛋白质保持可溶性时, 由于天门冬残留物对天冬氨酸24的去酰胺, 它的表面会产生负电荷, 这可能对其结晶有害。预测的 n-糖基化部位也可以突变, 最常见的是丙氨酸或谷氨酰胺残留物, 以防止这些部位的 n-链化糖基化, 并生成高均一性的糖蛋白样品。另外, 糖蛋白也可以在其他真核细胞细胞培养, 包括酵母, 昆虫, 植物系统, 或其他哺乳动物细胞系, 如中国仓鼠卵巢 (CHO) 细胞16,17。
许多哺乳动物表达载体, 包括 pHLsec, 允许分泌重组糖蛋白 ectodomains 进入细胞培养基25。分泌糖蛋白从 HEK293 细胞允许快速和容易的纯化, 不需要细胞裂解。添加纯化标签 (例如, 他的标签, 链球菌标记, 标志标记, c-myc 标记, HA 标记) 到目标糖蛋白的 N 或 C 终点, 允许单步亲和层析纯化。随后, 尺寸排除色谱可用于产生单分散样品的生物物理和结构表征。
在适当的条件下, 高纯度和均匀的糖蛋白样品可以产生良好的衍射晶体。一旦从这种晶体中获得了完整的 X 射线衍射数据集, 就需要确定初始阶段来计算糖蛋白的电子密度。由于蛋白质数据库 (PDB) 中的结构越来越多, 目前最常用的分阶段方法已经成为分子置换 (MR), 它使用相关的蛋白质结构来获得初始阶段26。然而, 当 MR 无法解决相位问题时, 就像偶尔出现的多域糖蛋白27、28、29的情况一样, 需要其他方法。本文详细介绍了用重原子 (HA) 浸泡晶体进行分阶段处理的方法, 为解决 CD22 胞外区28的结构要求。确定分阶段的右 HA 是一个迭代过程, 它取决于 ha 反应性、给定晶格中糖蛋白中的可用原子和结晶溶液30、31。另外, 在半胱氨酸和蛋氨酸残留物中的天然硫原子, 如果存在于糖蛋白中其他原子的足够高的比例, 则可以用于分阶段, 如果 X 射线衍射数据可以收集足够高的冗余32, 33。
膜糖蛋白的生物学功能通常由蛋白质相互作用或蛋白质配体相互作用 (如碳水化合物) 介导。当配体小到可以从溶液中扩散到晶体晶格中的糖蛋白结合部位时, 浸泡实验可以成功获得一个糖蛋白-配体共晶结构, 以更好地理解配体识别。
这里提出的协议也适用于了解表面糖蛋白与合成治疗配体34,35和抗体疗法36,37之间的相互作用。结合结构信息, 结合动力学和热力学可以有力地理解和改进它们的作用机制。一种允许对糖蛋白结合的治疗性抗体进行动力学分析的技术是 BLI38,39。BLI 使用生物传感器与固定化配体测量关联和离解动力学与一个结合伙伴, 最终确定平衡离解常数 (KD)。BLI 是一个有吸引力的方法, 因为需要少量的糖蛋白 (< 100 µg), 实验时间快 (每运行10-15 分钟), 它可以自动化。贸易中心对于研究糖蛋白和结合伙伴40、41、42、43之间的亲和力也很有用。虽然贸易中心是更多的时间和试剂密集型, 有价值的信息可以获得关于热力学的相互作用 (ΔG, ΔH, ΔS 和化学计量)。贸易中心对于研究与表面糖蛋白与配体的瞬态结合通常相关的弱相互作用也非常有用。此外, 这些技术可以结合使用, 以评估各种结构的结合, 并评估不同的 n-链接 glycoforms 表达糖蛋白在不同的细胞线的效果。执行 BLI 和贸易中心的糖蛋白产生的 HEK293F, HEK293S 和治疗与内 H 可以提供一个深入的看法多糖在生物活动和治疗参与的作用。
我们成功地应用这些协议来表征人类 CD2228的胞外领域 (幼儿发展), 这是唾液酸结合的类似凝集素 (Siglecs) 家族的糖蛋白成员, 对于维持 B 细胞稳态是必不可少的44.我们进行了深入的构造设计, 以促进结晶和分阶段的 X 射线数据集浸泡汞。我们还浸泡 CD22 晶体与配体唾液酸 (α2-6 唾液乳糖), 以获得免疫受体-配体复合体的结构, 从而提供了结构导向设计的糖 mimetics45,46的蓝图。此外, 我们产生了片段抗原结合 (anti-CD22 治疗抗体 epratuzumab-一个治疗候选者目前在 III. 期临床试验的非霍奇金淋巴瘤47-以确定其结合亲和性的 BLI 和贸易中心对差异糖化 CD22 幼儿发展的构造。这些研究揭示了 n-联用糖基化在 epratuzumab 参与中的关键作用, 对功能失调的 B 细胞 CD22 识别有潜在的影响。

<table border="0" cellpadding="0" cellspacing="0" style="width:860px;" width="859">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">0.22 &#956;m Steritop filter</td>
			<td style="width:163px;">EMD Millipore</td>
			<td style="width:155px;">SCGPS02RE</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 well 4-15% gradient SDS-PAGE gel</td>
			<td>Bio-Rad</td>
			<td>4561084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">10x glycobuffer 3</td>
			<td style="width:163px;">New England Biolabs</td>
			<td style="width:155px;">P0702S</td>
			<td>Comes with Endo H reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">10x Kinetics Buffer</td>
			<td>PALL Fort&#233;Bio</td>
			<td style="width:155px;">18-1092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">1 mL round bottom 96 well block</td>
			<td style="width:163px;">ThermoFisher</td>
			<td style="width:155px;">260251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">22 mm cover slip</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR3-231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:345px;">96-3 well INTELLIPLATE&#160; low volume reservior</td>
			<td style="width:163px;">Art Robbins Instruments</td>
			<td style="width:155px;">102-0001-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgeI</td>
			<td>New England Biolabs</td>
			<td>R0552S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">&#196;KTA Pure</td>
			<td style="width:163px;">GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">&#196;KTA Start&#160;</td>
			<td style="width:163px;">GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Amicon Ultra 15 centrifugal filtration device 10KDa MWCO</td>
			<td style="width:163px;">Millipore</td>
			<td style="width:155px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Amicon Ultra 4 centrifugal filtration device 10KDa MWCO</td>
			<td style="width:163px;">Millipore</td>
			<td style="width:155px;">UFC801008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Auto-iTC200</td>
			<td style="width:163px;">Malvern</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axygen MaxyClear Snaplock 1.5 mL microtubes</td>
			<td>Fisher Scientific</td>
			<td>MCT150C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CryoLoop 18 x 0.05-0.1 mm</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR4-945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CryoLoop 18 x 0.1-0.2 mm</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR4-947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CryoLoop 18 x 0.2-0.3 mm</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR4-970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Dry Bath</td>
			<td>Bio-Rad</td>
			<td>1660562EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli DH5&#945;</td>
			<td>Invitrogen</td>
			<td>18258012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Endo H</td>
			<td style="width:163px;">New England Biolabs</td>
			<td style="width:155px;">P0702S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask (baffled base), polycarbonate, sterile, 500 mL, DuoCAP</td>
			<td>TriForest Labware</td>
			<td>FBC05000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask 125 mL (baffled base), polycarbonate, sterile, 125 mL with vented cap</td>
			<td>VWR</td>
			<td>89095-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Disposable sterile serological pipet, non-pyrogenic, 10 mL</td>
			<td>Greiner Bio-One</td>
			<td>607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Disposable sterile serological pipet, non-pyrogenic, 25 mL</td>
			<td>Greiner Bio-One</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Disposable sterile serological pipet, non-pyrogenic, 5 mL</td>
			<td>Greiner Bio-One</td>
			<td>606180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Disposable sterile serological pipet, non-pyrogenic, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>768180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FectoPRO DNA Transfection Reagent, Polyplus</td>
			<td>VWR</td>
			<td>10118-842</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Freestyle 293F cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>R79007</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freestyle Expression medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12338001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Heavy Atom Screens Au</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR2-444</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Heavy Atom Screens Hg</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR2-446</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Heavy Atom Screens M1</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR2-448</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Heavy Atom Screens M2</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR2-450</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Heavy Atom Screens Pt</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR2-442</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK 293S</td>
			<td>ATCC</td>
			<td>ATCC CRL-3022</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HisTrap Affinity Column</td>
			<td>GE Healthcare</td>
			<td>17525501</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">HiTrap KappaSelect Affinity Columns</td>
			<td style="width:163px;">GE Healthcare</td>
			<td style="width:155px;">17545811</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">HiTrap LambdaSelect Affinity Columns</td>
			<td style="width:163px;">GE Healthcare</td>
			<td style="width:155px;">17548211</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">MCSG-1 Crystal Screen 1.7 mL block</td>
			<td style="width:163px;">Anatrace</td>
			<td style="width:155px;">MCSG-1</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">MCSG-2 Crystal Screen 1.7 mL block</td>
			<td style="width:163px;">Anatrace</td>
			<td style="width:155px;">MCSG-2</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">MCSG-3 Crystal Screen 1.7 mL block</td>
			<td style="width:163px;">Anatrace</td>
			<td style="width:155px;">MCSG-3</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">MCSG-4 Crystal Screen 1.7 mL block</td>
			<td style="width:163px;">Anatrace</td>
			<td style="width:155px;">MCSG-4</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Mercuric chloride</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:155px;">1044170100</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Microplate, 96 well, polypropelene, flat bottom, black&#160;</td>
			<td style="width:163px;">Greiner Bio-One</td>
			<td style="width:155px;">655209</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Minstrel DT UV</td>
			<td style="width:163px;">Formulatrix</td>
			<td style="width:155px;"></td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multitron Pro shaker</td>
			<td>Infors HT</td>
			<td>MP25-TA-CO2HB</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:345px;">Nanodrop 2000/2000c Spectrophotometer</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:155px;">ND-2000</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Nanosep 3K Omega centrifugal device</td>
			<td style="width:163px;">PALL Life Science</td>
			<td style="width:155px;">OD003C33</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ni-NTA biosensors</td>
			<td>PALL Fort&#233;Bio</td>
			<td>18-5102</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Octet RED96</td>
			<td style="width:163px;">PALL ForteBio</td>
			<td style="width:155px;"></td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Oryx 4 crystallizaiton robot</td>
			<td style="width:163px;">Douglas Instrument</td>
			<td style="width:155px;">ORY-4/1</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Platinum chloride</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:155px;">520632-1g</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Plus Protein Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureLink HiPure Plasmid Maxiprep Kit</td>
			<td>Invitrogen</td>
			<td>K210006</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quick Coomassie Stain</td>
			<td>Protein Ark</td>
			<td>GEN-QC-STAIN-1L</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steriflip Sterile 50 mL Disposable Vacuum Filtration System 0.22 &#181;m Millipore Express</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Superdex 200 Increase 10/300 GL</td>
			<td style="width:163px;">GE Healthcare</td>
			<td style="width:155px;">28990944</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Superose 6 10/300 GL</td>
			<td style="width:163px;">GE Healthcare</td>
			<td style="width:155px;">17517201</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Tantalum bromide cluster</td>
			<td style="width:163px;">Jena bioscience</td>
			<td style="width:155px;">PK-103</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Top96 Crystallization Screen</td>
			<td style="width:163px;">Rigaku Reagents</td>
			<td style="width:155px;">1009846</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryphan Blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">VDX 24-well with sealant</td>
			<td style="width:163px;">Hampton research</td>
			<td style="width:155px;">HR3-172</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">&#945;2-6 sialyllactose</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:155px;">A8556-1mg</td>
			<td style="width:197px;"></td>
		</tr>
	</tbody>
</table>

characterization of glycoproteins with the immunoglobulin fold by x-ray crystallography and biophysical techniques

细胞表面的糖蛋白在细胞功能中起重要作用, 包括信号、黏附力和转运。在白细胞上, 这些糖蛋白中有几个具有免疫球蛋白 (Ig) 褶皱, 并且是免疫力识别和调节的核心。在这里, 我们提出了一个平台的设计, 表达和生物物理特性的细胞外领域的人 B 细胞受体 CD22。我们建议, 这些方法是广泛适用于哺乳动物糖蛋白 ectodomains 的特点, 其中含有的免疫球蛋白领域。两个悬浮人胚肾 (HEK) 细胞系, HEK293F 和 HEK293S, 是用来表达的糖蛋白窝藏复杂和高甘露糖多糖分别。这些重组糖蛋白与不同的 glycoforms 允许研究糖大小和组成对配体结合的影响。我们讨论了研究糖蛋白结合生物学相关配体和治疗抗体候选者的动力学和热力学的协议。HEK293S 细胞生成的重组糖蛋白, 由于糖的均一性, 降低了灵活性和 endoglycosidase H 治疗的易感性, 易于结晶。我们提出了用重原子和小分子浸泡糖蛋白晶体的方法来测定和分析配体结合。这里讨论的实验性协议对哺乳动物糖蛋白的特性有一定的把握, 可以洞察它们的功能, 并研究治疗的作用机制。

在哺乳动物 HEK293F 和 HEK293S 细胞株中成功地克隆出了 CD22 的几种结构, pHLsec 表达载体和抗原 (图 2和 3A)。通过尺寸排除色谱法纯化所有构造, 并产生高度纯净的结晶研究样品 (图 3B和 3C)。导致衍射晶体的 CD22 构造是 d1-d3 截断 (残滓 20-330), 其中六预测的 n-链化的糖基化部位从 Asn 到亚拉巴马州 (N67A、N112A、N135A、N164A 和 N231A), 在 HEK293S 细胞中产生, 只有在位置 N101 的糖基化站点被保留了 (这个结构命名 CD2220-330, 5A)。晶体是在 MCSG-1 稀疏矩阵的几个条件, 但最好的晶体是从一个条件, 包括 30% (w/v) 聚乙二醇 4000, 0.2 米氯化锂和0.1 米三, pH 8.5。这些原生晶体衍射到2.1 Å分辨率;使用已知结构的 Siglec 相关蛋白质的玻璃领域没有产生任何解决方案在 MR 搜索。
为了获得分阶段的信息, 我们用一个 ha 化合物的面板浸泡了本机晶体, 其中包括汞、Pt、Os、Ta 和 Br, 其浓度从1-20 毫米 ha 化合物到孵化时间从5分钟到 1 d 不等 (图 4)。我们监测晶体的形态学变化, 发现晶体浸泡在20毫米的 HA 化合物导致快速开裂和溶解晶体。我们冻结了63种晶体, 在设定的孵化时间后保持其形状, 它们浸泡在溴化钽簇、铂氯、醋酸汞和氯化汞中。用7毫米氯化汞浸泡的晶体30分钟在加拿大光源 (CLS) 08-BM 光束线 (加拿大萨斯卡通) 的荧光扫描中显示异常信号, 允许在单个晶体。这些数据集使我们能够解决 CD2220-330 5A的汞子结构, 它揭示了一个单一的汞原子, 它被绑定到一个自由的半胱氨酸位置 C308, 最终允许我们建立 CD2220-330, 5A的结构到分阶段电子密度图使用 AutoBuild78。
一旦 unliganded 结构得到解决, 我们就有兴趣解决 CD22 与配体、α2-6 siallylactose 的结构。我们首先计算了 CD22 对α2-6 唾液乳糖的亲和性, 利用贸易中心来表征相互作用的结合热力学。我们观察到了280µM 的亲和性, 并利用这些信息来确定配体的初始浓度 (~ 100 x KD) 用于浸泡我们的本地 CD2220-330,5A晶体。我们浸泡的 CD2220-330, 5A晶体与25毫米 siallylactose 5 分钟, 2 h, 14 h, 40 h 和 5 d 和监测的变化, 晶体形态。共75个晶体被冻结从不同的时间点, 并发送到 CLS 同步光束线 08 ID (加拿大萨斯卡通), 为远程数据收集。从衍射晶体中收集了总共六个 x 射线数据集。利用 unliganded CD2220-330 5A结构作为初始搜索模型, 解决了每个 X 射线数据集的结构。然后, 对所有数据集产生的电子密度进行检查, 以确定在α2-6 地图中的正密度, 该映射对应于 CD22 绑定站点内的绑定唾液乳糖。值得注意的是, 所有收集到的数据集, 即使是在5分钟的潜伏期内浸泡的晶体, 也含有与结合部位对应的配体的正密度。unliganded 和 liganded CD22 的整体结构与最小构象变化高度相似, 这可能解释了α2-6 唾液乳糖浸泡实验的成功与否。
我们接下来的特点是在 BLI 和国贸中心的实验中承认的 CD22 抗原表面 epratuzumab (图 5)。epratuzumab 的动力学和热力学剖面结合到 CD22 构造与不同的 glycoforms 揭示了越来越亲和 CD22 与减少 N 链接糖大小, 多达14倍的改善亲和力为较小的多糖 (327 毫微米 vs 24 nm在 BLI;188毫微米 vs 58 毫微米在贸易中心)。采用 CD22 单点突变体 BLI, 通过求解 epratuzumab Fab-CD22 共晶结构28, CD22 N 联糖限制了抗体对其表位的获取。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57750/57750fig1.jpg"> 图 1.从结构设计到生物物理和结构表征的糖蛋白特征概述.(1) 代表性糖蛋白的初步序列分析。在灰色, 胞外领域 (幼儿发展);绿色, 跨膜 (TM) 段;在蓝色中, 糖蛋白的胞浆域。预测的 N 链接多糖被标记。(2) 幼儿发展的克隆。(3) 哺乳动物细胞的早期发育表达。(4) 糖蛋白纯化。在 HEK293F 中表达的蛋白质将含有复杂的多糖, HEK293S 中表达的蛋白质将有高甘露糖多糖。酶法处理 HEK293S 细胞中产生的糖蛋白, 其结果是在 n-链接糖基化部位只有一个 GlcNAc 基团的糖蛋白。(5a) 糖蛋白通过 biolayer 干涉测量 (BLI) 和等温滴定量热仪 (贸易中心) 对抗体的结合进行测试。与小配体的亲和性也可以通过贸易中心来衡量。(5b) 糖蛋白与均匀 n-链多糖的结晶试验, 如在 HEK293S 和 deglycosylated 中表达的与内 H. (6) 在某些情况下, N 链接糖基化部位的突变是获得晶体所必需的。<a href="https://www.jove.com/files/ftp_upload/57750/57750fig1large.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/57750/57750fig2.jpg"> 图 2.CD22 胞外区 DNA 构建在哺乳动物细胞中表达的设计. A) pHLsec 质粒的表达, 用于瞬态转染 CD22 的早期发育。用于克隆的 AgeI 和 KpnI 站点用红色框表示。B) CD22 的幼儿发育包括七个 d1-d7 和12预测的 n-链化的糖基糖化点 (蓝色)。四构造是从 CD22 幼儿开发中设计的。C) 1% 琼脂糖凝胶显示 PCR amplicons 的 CD22 幼儿早期构建克隆成 pHLsec 哺乳动物表达载体。第一条车道包含 1 kb DNA 标记。<a href="https://www.jove.com/files/ftp_upload/57750/57750fig2large.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/57750/57750fig3.jpg"> 图 3.糖蛋白的表达和纯化。A)细胞密度对表达率的影响。小尺度25毫升培养的糖蛋白表达 HEK293F 悬浮细胞的三种不同起始密度细胞 (0.5 x 106细胞 ml-1, 1.0 x 106细胞 ml-1, 1.5 x 106细胞毫升-1)。从左面板中的 SDS 页和在右面板中定量 BLI 的密度测量进行量化。值代表一个糖蛋白制剂。B)第一纯化步骤的色谱, 用于构造 CD2220-330, 5A从600毫升的上清, 使用镍 NTA 亲和柱。糖蛋白被洗脱使用咪唑 (灰色线) 梯度, 其中100% 对应于洗脱缓冲, 其中包含500毫米咪唑。汇总的分数用垂直线描述。C)使用高性能凝胶过滤柱构造 CD2220-330,5A的尺寸排除色谱。从洗脱峰中汇集的分数用垂直线描述。插图: 考马斯染色的 SDS 页凝胶显示糖蛋白的纯度。<a href="https://www.jove.com/files/ftp_upload/57750/57750fig3large.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/57750/57750fig4.jpg"> 图 4.用重原子浸泡的晶体.A) 用 HA 化合物浸泡本机晶体的样品工作站。所有必需的工具都被标记。B) 采取步骤, 浸泡晶体的建筑 CD2220-330, 5A与 HA 化合物。步骤 1, 打开含有良好的晶体, 并转移晶体使用一个环到0.2 µL 下降的封面滑动包含 HA 溶液稀释在结晶条件, 使 ha 的最终浓度从1-10 毫米不等。步骤 2, 密封在结晶板的下降和孵化晶体与 HA 化合物在不同的时间段。步骤 3, 在循环中装入浸泡过的水晶, 并在三连续0.2 µl 滴中浸泡三十年代, 其中含有母液溶液, 辅以 20% (v/v) 甘油, 并将其放在盖子上。步骤 4, 闪光冻结的水晶安装在一个循环与液氮, 并把它放在一个冰球运往同步辐射光束线。<a href="https://www.jove.com/files/ftp_upload/57750/57750fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57750/57750fig5.jpg"> 图 5.Biolayer 干涉测量和等温滴定量热测定。A)代表 BLI 实验。顶部面板:一个动力学实验的平板设置示例: 1x 动力学缓冲器 (B), 他的6x标签糖蛋白加载 (L), 代表性的晶圆浓度 (500, 250, 125, 62.5 nM), PBS + 500 毫米再生缓冲器 (R) 和1x 动能中和缓冲器 (B)。每个井包含200µL 的解决方案。动力学实验的步数在板材的顶部被表明。中间板:BLI 实验的代表性原始数据使用 Ni-NTA 生物传感器和板块描述的顶部板。步数对应于基线 (1), 他的6x糖蛋白装载 (2), 基线 (3), 协会在连续稀释的晶圆 (4) 和离解 (5)。未表示再生步骤 (步骤 6-7)。底部面板:代表分析的数据显示原始的关联和离解 (蓝线) 与相应的1:1 适合 (红线)。B) 顶部面板:一个单一的贸易中心在一个96井的圆底块上进行七次实验的自动化贸易中心仪器上运行的代表性板设置。每个实验由三口井组成。第一井 (红色) 对应于样品为细胞 (400 µL), 第二个井 (绿色) 对应于样品为注射器 (120 µL)。第三井是空的, 混合样本将返回到这个良好的实验完成后。实验1、2和7是缓冲控制的缓冲区。实验3-5 代表细胞中的糖蛋白 (P) 的三项实验和在注射器中的晶胞或配体 (L)。实验6代表稀释控制的配体热, 在数据分析过程中应从实验3-5 中减去。底部面板:代表性的原始 (顶部) 和处理 (底部) 贸易中心数据显示, epratuzumab 对 CD22 在 HEK293F 细胞中产生的发育具有约束力。<a href="https://www.jove.com/files/ftp_upload/57750/57750fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques at JoVE.com

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

通过 biolayer 干涉法、等温滴定量热仪和 X 射线晶体学, 我们提出了免疫球蛋白褶皱的生物物理和结构表征方法。

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

characterization-glycoproteins-with-immunoglobulin-fold-x-ray

Research

Education

JoVE Journal

JoVE Core

Chemistry

Biochemistry

Liquids, Solids, and Intermolecular Forces

SCIENTISTS IN ACTION

12.5K 次观看.  The Hospital for Sick Children Research Institute. 通过 biolayer 干涉法、等温滴定量热仪和 X 射线晶体学, 我们提出了免疫球蛋白褶皱的生物物理和结构表征方法。

视频: Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

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

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

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

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

小角度 x 射线散射溶液中大分子的结构研究

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

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 ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

优化用于连续晶体学实验的内硫肽晶体的生长

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...