Type of Document Dissertation Author Russo, Cristina URN etd-05062010-184102 Title Salt-Dependence of Protein-DNA Binding: Insights into Protein Electrostatics and Redesign Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title Marcia O. Fenley Committee Chair Michael Blaber Committee Co-Chair Hugh Nymeyer Committee Member Timothy Logan Committee Member Michael Mascagni University Representative Keywords
- DNA-Binding Proteins
- Protein Design
Date of Defense 2010-05-03 Availability unrestricted AbstractElectrostatics interactions are fundamental for protein stability and function, and play a crucial role upon protein-DNA binding. Both the protein and the DNA from a complex exhibit clear shape and electrostatic complementarity. Counterions from the solvent surround the DNA in order to neutralize its highly anionic charges. The interplay between the delocalized binding and release of counterions has been originally established by the counterion condensation theory (CCT). The correlation between the binding constant Kobs and the salt concentration [M+] provides the measurement SKobs. CCT also states that the number of ionic contacts formed in the interface of protein and DNA during binding equals SKobs.
In this work we explore the validity of the above relationship and we question the efficacy of SKobs descriptions for larger, more complex protein-DNA systems. We also analyzed the role of the charge distribution in protein stability and potentially in binding affinity between
protein and DNA. We employ a computational approach that accounts for the non-specific salt-mediated electrostatic interactions by measuring the electrostatic free energy of binding with the Poisson-Boltzmann (PB) equation. We investigated the distinct salt-dependent binding behavior of halophilic TATA-binding proteins (TBP) to DNA and of four families of DNA-binding proteins (homeodomains, highmobility
groups, interferon regulatory factors, and basic-region leucine zippers). We were able to obtain a PB-based definition of SK for the protein-DNA complexes (SKpred), which compares to SKobs, except in cases when protein and/or DNA undergo dramatic conformational changes.
We did not observe the previously determined correlation between SK and ion pairs devised by CCT for any of the protein-DNA complexes in this study, and therefore we believe this correlation does not hold for complexes larger than the original oligocationpolyelctrolytes tested. Since the correlation was determined for a series of oligocationpolyelectrolyte complexes, where the DNA was represented by a short line of charges (instead of the double stranded conformation of DNA), the correlation fails to capture the molecule's structural information.
Besides, the correlation between SK and ion pairs only accounts for protein cations as ion pair forming. For that reason, we decided to test the effect of anions over SKpred. By neutralizing the charge of anionic residues at the binding interface of TBP and of the other proteins of the four DNA-binding protein families, we noticed a severely altered SKpred. In some cases, we performed surface mutations on TBP, in order to reverse the charge of anionic residues at TBP's binding interface, in which case the halophilic nature of the protein approaches a mesophilic-like behavior when bound to DNA. Combined, these results indicate the crucial effect of charge distribution into SKpred and potentially to the the stability of a protein-DNA complex.
Our studies led to the development a protein redesign program that can automatically generate protein mutants, in an effort to achieve protein structures with higher thermal stability. We created a de novo protein design algorithm based on a Monte Carlo method coupled with a replica exchange approach. We validated the usefulness of our approach in repacking a large group of diverse proteins and also compared the effect of different parameters (such as force field and rotamer libraries) upon the final resulting structures. We observed that our approach is system-independent, and still eficient for very large proteins. Our algorithm was employed in the modeling of more stable variants of fibroblast growth
Based on the work presented here, we believe that current interpretations of SKobs will have to be reconsidered, and we are currently working to expand CCT. We observed that the effects from the charge distribution are much more complex and can relate to protein stability, as seen in the thermophilic TBP. The charged mutations obtained from our protein design algorithm could help to engineer proteins with higher thermostability. Newly redesigned proteins could potentially be more resistant to degradation or have longer shelf-life; therefore aiding the fields of industry and medicine.
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