Assessing a potential correlation between protein stability and function of the HMG domain of Lymphoid Enhancer Binding Factor-1

Lymphoid enhancer binding factor-1 (LEF-1) is an architectural transcription factor that is a member of the HMGB family. The NMR spectra of the LEF-1 DNA binding domain (HMG domain) complexed with DNA exhibits the characteristic NMR spectra of a folded globular protein. In contrast, NMR analysis of the free LEF-1 HMG domain revealed the presence of significant structural instability and conformational heterogeneity. Its disorder-to-order transition upon binding to DNA renders LEF-1 an excellent candidate for exploring the potential role stability plays in the function of the HMG domain. To gain insight into the possible role of LEF-1 disorder, or lack of stability prior to binding to DNA, we undertook the challenge of introducing stability into the HMG domain of LEF-1. This work will first demonstrate the use of homology modeling, structural analysis, and protein design algorithms (ORBIT) in an attempt to stabilize the HMG domain of LEF-1. Nine variants of the HMG domain of LEF-1 were created and structurally analyzed using NMR, CD, and limited proteolysis. Five variants were reported to be more stable than wild type. Lastly, we assessed the degree by which the nine mutant variants function in binding and bending cognate DNA. The results indicate that increased stability does not affect the degree by which the LEF-1 domain bends DNA but does have a small yet consistent affect on the binding affinity.