TELSAM polymers are powerful crystallization chaperones meriting continued investigation

TELSAM 聚合物是强大的结晶伴侣，值得继续研究

• 批准号: 10438316
• 负责人: James Daniel Moody
• 金额: $ 42.91万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10438316
• 关键词: ANTXR2 gene; Address; Adopted; Adoption; Biotechnology; Cryoelectron Microscopy; Crystallization; Crystallography; Development; Disease; ETV6 gene; Goals; Health Care Costs; Human; Investigation; Left; Length; Ligands; Malignant Neoplasms; Mediating; Methods; Mission; Molecular; Molecular Chaperones; Molecular Disease; National Institute of General Medical Sciences; Organism; Polymers; Process; Proteins; Public Health; Research; Research Project Grants; Resolution; Resources; Roentgen Rays; SAM Domain; Set protein; Side; Speed; Structure; Techniques; Testing; Time; United States National Institutes of Health; Variant; cost; effective therapy; experimental study; flexibility; human disease; innovation; interest; leukemia; new technology; novel strategies; programs; protein complex; protein function; protein structure; success; symptom management; tool

There is a critical need for new protein crystallization methods that require less labor, time, and resources. Pre- viously, crystals of 10 out of 11 target proteins were readily generated by fusing them to TELSAM, a polymer- forming crystallization chaperone. There is great need for continued investigation of TELSAM due to its potential as a general-use protein crystallization chaperone. Lack of straightforward methods to successfully crystallize any protein of interest significantly hinders study of molecular disease mechanisms and the development of effective treatments. The lack of effective treatments for many diseases forces them to be addressed instead with costly symptom management programs. The long-term goal of this project is to develop protein crystalliza- tion methods that can result in well-ordered protein crystals on a time scale of less than a month, cost as little as $1000 per structure, and are successful for greater than 70% of proteins of interest. The overall objective of this proposal is to convincingly demonstrate the benefits of using TELSAM as a protein crystallization chaperone and to clearly define the requirements for doing so. The central hypothesis is that TELSAM will accelerate the speed and success rate of crystallization across a wide range of proteins of interest and that flexible fusion of target proteins to the 1TEL variant will be optimal. The rationale is that TELSAM has shown great promise in preliminary studies and has the potential to 1) decrease the cost of determining an atomic-scale protein structure, 2) accel- erate the rate that protein structures can be determined, and 3) increase the success rate of crystallization, expanding the range of proteins that can be structurally characterized in this way. The central hypothesis will be tested, and the overall objective achieved by executing 2 specific aims: 1) Compare the ease of obtaining well- ordered crystals across a range of proteins of interest with and without fusion to TELSAM. 2) Establish best practices for successfully using TELSAM. In Aim 1, a panel of target proteins or protein complexes of varying sizes will be crystallized alone or as flexible fusions to TELSAM. In Aim 2, selected target proteins will be flexibly or rigidly fused to TELSAM with varying degrees of target protein loading along the polymer. Longer linker lengths and unusually low protein concentrations in crystallization experiments will also be investigated. The proposed research is innovative, in the applicant’s opinion, because it proposes: 1) Systematic investigation of the factors required by TELSAM-target fusions to reliably form well-ordered crystals, 2) Investigation of 1TEL, which pre- sents 6 copies of the target protein per turn of the TELSAM polymer and precludes any direct inter-TELSAM contacts, 3) Investigation of semi-rigid fusions of target proteins to TELSAM, 4) Testing the limits of TELSAM- mediated protein crystallization with target protein complexes and ligand-bound targets. The proposed research is significant because it will enable the successful crystallization and structure determination of a greater number and variety of biotechnology and disease-relevant proteins, ultimately leading to new biotechnology tools, more effective disease treatments, and reduced healthcare costs.

迫切需要更少劳动力、时间和资源的新的蛋白质结晶方法。预处理 然而，11种靶蛋白中有10种的晶体很容易通过将它们融合到TELSAM中产生，TELSAM是一种聚合物， 形成结晶伴侣。由于TELSAM的潜力，非常需要继续对其进行研究 作为一种通用的蛋白质结晶伴侣。缺乏直接的方法来成功地结晶 任何感兴趣的蛋白质都显著地阻碍了分子疾病机制的研究和 有效的治疗。许多疾病缺乏有效的治疗方法，迫使人们转而关注这些疾病。 昂贵的症状管理项目。该项目的长期目标是开发蛋白质结晶- 可以在不到一个月的时间范围内产生有序蛋白质晶体的方法，成本低至 每个结构1000美元，并且成功用于超过70%的感兴趣蛋白质。本报告的总体目标 该提案令人信服地证明了使用TELSAM作为蛋白质结晶伴侣的益处， 明确规定这样做的要求。核心假设是，TELSAM将加速 和跨广泛范围的感兴趣蛋白质的结晶的成功率，以及靶的柔性融合 将蛋白质与1 TEL变体结合将是最佳的。理由是，TELSAM在初步研究中显示出了巨大的潜力， 研究并有可能1）降低确定原子尺度蛋白质结构的成本，2）加速 提高了蛋白质结构的确定率，3）提高了结晶的成功率， 扩大了可以用这种方式进行结构表征的蛋白质的范围。核心假设是 测试，并通过执行2个具体目标实现的总体目标：1）比较获得良好- 在与和不与TELSAM融合的情况下，在一系列感兴趣的蛋白质上的有序晶体。2)确立最佳 成功使用TELSAM的实践。在目标1中，一组不同的靶蛋白或蛋白复合物被用于靶蛋白或蛋白复合物。 尺寸将单独结晶或作为TELSAM的柔性融合物。在目标2中，选择的靶蛋白将被灵活地 或刚性融合到TELSAM上，沿着聚合物沿着具有不同程度的靶蛋白负载。更长的接头长度 并且还将研究结晶实验中异常低的蛋白质浓度。拟议 申请人认为，这项研究是创新的，因为它提出：1）系统地调查各种因素， 所需的TELSAM目标融合，以可靠地形成良好有序的晶体，2）调查的1 TEL，这预- 每转TELSAM聚合物发送6个拷贝的靶蛋白，并排除任何直接的TELSAM间 接触，3）研究靶蛋白与TELSAM的半刚性融合，4）测试TELSAM的极限- 介导的蛋白质结晶与靶蛋白复合物和配体结合的目标。拟议研究 是重要的，因为它将使更多的成功结晶和结构确定 以及各种生物技术和疾病相关蛋白质，最终导致新的生物技术工具， 有效的疾病治疗，降低医疗成本。

项目成果

Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.

降低 TELSAM 目标蛋白接头的灵活性并省略可切割的融合标签可改善晶体顺序和衍射极限。

• DOI: 10.1101/2023.05.12.540586
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Gajjar,ParagL;Romo,MariaJPedroza;Litchfield,CelesteM;Callahan,Miles;Redd,Nathan;Nawarathnage,Supeshala;Soleimani,Sara;Averett,Jacob;Wilson,Elijah;Lewis,Andrew;Stewart,Cameron;Tseng,Yi-JieJ;Doukov,Tzanko;Lebedev,Andrey;Mood
• 通讯作者: Mood