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.

需要更少的人工，时间和资源需要新的蛋白质结晶方法。 pre 最明显的是，通过将11个靶蛋白的10个晶体融合到Telsam（一种聚合物）中，很容易产生。 形成结晶伴侣。由于其潜力，Telsam非常需要继续投资 作为通用蛋白质结晶伴侣。缺乏成功结晶的直接方法 任何有趣的蛋白质都显着阻碍分子疾病机制的研究和发展 有效的治疗方法。缺乏许多疾病的有效治疗迫使他们被解决 具有昂贵的症状管理计划。该项目的长期目标是开发蛋白质结晶 - 可以在少于一个月的时间范围内导致有序良好的蛋白质晶体的方法，费用不到 每个结构$ 1000，成功的蛋白质超过70％。总体目标 提案是令人信服地证明使用telsam作为蛋白质结晶链的好处 清楚地定义这样做的要求。中心假设是Telsam将加速速度 以及在广泛感兴趣的蛋白质中结晶的成功率和目标的灵活融合 1TEL变体的蛋白质将是最佳的。理由是Telsam在初步方面表现出了巨大的希望 研究并有可能1）降低确定原子级蛋白质结构的成本，2）加速 - 确定可以确定蛋白质结构的速率，3）增加结晶的成功率， 扩展可以以这种方式在结构上表征的蛋白质范围。中心假设将是 经过测试，以及执行2个具体目标实现的总体目标：1）比较获得良好的便利性 在与TELSAM融合的情况下有和不融合的一系列感兴趣的蛋白质中有序的晶体。 2）建立最好的 成功使用Telsam的实践。在AIM 1中，一组目标蛋白或蛋白质复合物的不同 尺寸将单独结晶，或与Telsam的柔性融合。在AIM 2中，选定的靶蛋白将是灵活的 或将沿聚合物的靶蛋白负荷变化的不同程度的靶蛋白载与TELSAM融合在一起。较长的接头长度 还将研究结晶实验中异常低的蛋白质浓度。提议 申请人认为，研究具有创新性，因为它的建议：1）系统投资这些因素 Telsam-target融合需要可靠地形成有序的晶体，2）研究1TEL，这是 每回合TELSAM聚合物每回合发送6份靶蛋白，并排除任何直接telsam 接触，3）研究靶蛋白与Telsam的半刚性融合的研究，4）测试Telsam-的极限 介导的蛋白质结晶与靶蛋白络合物和配体结合靶标。拟议的研究 之所以重要，是因为它将能够成功的结晶和结构确定更多 以及各种生物技术和与疾病相关的蛋白质，最终导​​致新的生物技术工具，更多 有效的疾病治疗和降低医疗费用。

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