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使用尾酒浸泡和自动化MicroED的连接物查和发现

Jieye Lin¹, Marc J Gallenito¹, Johan Hattne^1,2

¹Department of Biological Chemistry, University of California, Los Angeles, 615 Charles E. Young Drive South, Los Angeles, California 90095, United States.

bioRxiv : the preprint server for biology

|March 3, 2025

概括

这项研究引入了一种新的工作流程,将尾酒浸泡与微晶电子衍射 (MicroED) 结合起来,以快速选蛋白质-连接物和从微晶体中确定结构,提高命中率和结合分析.

科学领域:

结构生物学结构生物学
生物化学生物化学
晶体学晶体学是指结晶学.

背景情况:

传统上使用单晶X射线衍射 (SC-XRD) 来对蛋白质-连接体相互作用进行高通量晶体学选.
这种方法对于蛋白质微晶来说是有限的,因为它们太小,无法产生强烈的衍射模式.

研究的目的:

开发一个集成的工作流程,用于从蛋白质微晶体直接快速选连接体,结构确定和结合分析.
克服SC-XRD在结构生物学中的小微晶体的局限性.

主要方法:

尾酒浸泡技术与自动微晶电子衍射 (MicroED) 的整合.
工作流的验证,使用已知用于热溶解的配体.
应用用于识别蛋白酶K联体的新型结合相互作用.

主要成果:

成功的快速连接体选,结构确定和直接从微晶体进行结合分析.
对蛋白酶K.的新型结合相互作用的鉴定.
多个蛋白质-连接体复合体的结构,包括具有较弱结合亲和力的结构,得到了高效的解决.
估计的相对结合亲和度与先前的研究和微量热泳 (MST) 测量结果相对应得很好.

结论:

开发的工作流程使微晶体中的蛋白质 - 连接体复合物的高效结构分析成为可能.
这种方法提高了连接体选的命中率,并提供了对连接相互作用的洞察力,即使是对弱结合的连接体.
该方法是结构生物学和药物发现工作的宝贵工具.

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