Structure and Selectivity of Designed Metallohomeodomains

University Of Iowa, Iowa City IA

Investigators

Abstract

The focus of this work is to incorporate a series of metal-binding loops into a protein domain, utilizing a modular approach to protein design in which isostructural metal-binding loops are substituted into a helix-turn-helix (HTH) DNA-binding domain. The long-term goal is to engineer novel metalloproteins with tunable hydrolytic activity and DNA sequence selectivity. This work is a foundation for understanding protein-DNA recognition and the contextual reactivity of metal-binding loops generally. Specifically, a series of chimeric proteins will be prepared and characterized to elucidate key design features involved in protein stability and DNA sequence selectivity. The following three aims will be addressed: 1) Incorporate an EF-hand metal-binding site into a HTH domain, and test the optimal size of the turn mutation, the importance of hydrophobic residues flanking the EF-hand, and the effect of the EF-hand loop sequence on Ln-binding and domain folding. 2) Determine the affinity and reactivity of the metallo-homeodomains toward DNA, as a function of loop position and metal binding. 3) Tune DNA-binding selectivity and affinity by domain dimerization, by modularly combining recognition units to yield new specificity. A series of chimeric engrailed dimers will be prepared, comparing linker length and relative orientation of the domains (head-to-head (disulfide linkers) vs. head-to-tail with varying poly-glycine linkers). This research represents a new biomimetic strategy for investigating metalloprotein-nucleic acid interactions. This chimeric approach is unique in that it addresses two functions synergistically (catalysis and sequence targeting), and tests the underlying principles governing recognition with spectroscopically rich, hydrolytically active small constructs. Designing an enzyme from first principles that actually folds and has biological activity serves as a benchmark of our understanding of protein folding, metalloenzyme active-site reactivity, and the many subtle factors involved in tuning recognition to yield the incredible diversity of function seen in nature. Broader Impacts. These studies rely on a diverse, interdisciplinary experimental approach, which offers students and postdoctoral fellows a multifaceted training experience. This project provides training in areas ranging from molecular biology to physical measurements to spectroscopy. Furthermore, students at a local college (Loras College, Dubuque, IA) will utilize these chimeras in developing discovery-based Biochemistry labs for upper-level undergraduates, based on specific point mutations, protein expression, and NMR characterization of the metallohomeodomains. The engineered enzymes could potentially have diverse biotechnological applications.