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

Baylor University, Waco TX

Investigators

Abstract

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.