Direct Thermodynamic Quantification of Single-strand DNA Binding Proteins Cooperativities and Conformations

有了这个奖项，化学部的生命过程化学计划正在资助迈克尔·A。贝勒大学的Trakselis研究蛋白质如何与单链DNA结合。细胞的遗传信息存储在DNA中，但为了读取这些信息，必须在单链DNA结合蛋白（SSB）的帮助下解开并稳定这种双螺旋分子。该项目使用一种称为天然质谱（nMS）的方法来非常准确地确定SSB结合的单链DNA的大小，然后显示SSB在组装成这样的DNA复合物时是否以合作的方式结合。当与其他生物物理方法的结果相结合时，这些研究将有助于阐明来自不同生物体的SSB与单链DNA结合并可能改变其形状的机制。从这个项目中获得的基础知识预计将提供更好的理解如何在DNA中的信息复制和修复时发生错误。此外，成功使用nMS研究SSB与DNA的结合将有助于开发其应用于研究其他蛋白质组装成生物学上重要的功能复合物的方法，包括一种组分的结合如何以合作的方式刺激下一种组分的结合。该项目包括科学推广计划，旨在鼓励当地小学生通过“DNA日”探索核酸科学的奇迹，通过当地农贸市场的科学日参与社区活动，并通过“展示你的博士学位”演讲鼓励当地高中生在大学里追求科学。来自不同来源的单链结合蛋白（SSB）将通过先进的MS和荧光方法用于探测和量化协同结合的热力学和构象。SSB以单体形式和寡聚复合物存在;具有不同的结合模式，包括从非合作到强合作，并且在结合时，具有改变DNA构象的能力。一些SSB的热力学和结构性质已经被彻底表征，而其他SSB的结合性质则更加矛盾，并且不太好表征。该项目将利用不同类别的SSB来验证nMS方法在定义明确的系统上的应用，揭示其他新型SSB的结合模式和性质，并为其他人直接量化结合自由能和结构表征大分子组装中的SSB提供框架。由此产生的数据、方法和发现将通过多个渠道在科学界广泛传播，并有望为SSB绑定的协同性提供热力学见解。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Michael A. Trakselis from Baylor University to study how proteins bind to single-stranded DNAs. The cell’s genetic information is stored in DNA, but in order to read this information, this double helical molecule must be unwound and stabilized with the help of single-strand DNA binding proteins (SSBs). This project uses a method known as native mass spectrometry (nMS) to very accurately determine the size of single-stranded DNAs bound by SSBs, which then shows whether SSBs bind in a cooperative manner when assembling into such DNA complexes. When combined with results from other biophysical methods, these studies will help elucidate the mechanisms by which SSBs from different organisms bind to single-stranded DNAs and potentially change their shapes. Fundamental knowledge gained from this project is expected provide a better understanding for how the information in DNA is duplicated and repaired when errors occur. In addition, the successful use of nMS to study SSB binding to DNAs will help develop the method for its application to study the assembly of other proteins into biologically important functional complexes, including how the binding of one component stimulates the binding of the next in a cooperative manner. This project includes scientific outreach programs designed to encourage local elementary school students to explore the wonders of nucleic acid science through "DNA Days", to engage the community though Science Days at local Farmer’s markets, and to encourage local high school students to pursue science in college through “Present Your PhD” presentations.In this project, single-stranded binding proteins (SSBs) from diverse sources will be used to probe and quantify the thermodynamics and conformations of cooperative binding through advanced MS and fluorescence approaches. SSBs exist in both monomeric form and oligomeric complexes; have varied binding modes including from non-cooperative to strongly cooperative, and upon binding, have the ability to alter DNA conformation. The thermodynamic and structural properties of some SSBs have been thoroughly characterized, while the binding properties of other SSBs are more contradictory and less well-characterized. This project will utilize diverse classes of SSBs to validate the application of nMS methods on well-defined systems, reveal binding modes and properties for other novel SSBs, and provide a framework for others to directly quantify free energies of binding and to structurally characterize SSBs in macromolecular assemblies. The resulting data, methods, and findings will be broadly distributed within the scientific community through multiple outlets and are expected to provide thermodynamic insight into cooperativity of SSB binding.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.