Nature controls the chemical reactivity of metalloproteins by varying the number, identity, and geometry of the coordinating amino acids in the protein active site. Our current understanding of how and why these variations lead to functional changes is rather limited, significantly inhibiting progress in the field of molecular medicine. The overall objective of this project is to understand the structure-function relationships in di-iron carboxylate enzymes, which catalyze a diversity of biologically important chemical reactions despite considerable similarities in their active site configurations. To accomplish this goal, we will computationally design, experimentally produce, and comprehensively characterize a series of small, unnatural model proteins in which the number, identity, and geometry of the iron- coordinating amino acids are systematically varied. Our scaffold of choice is DFsc, a member of the due ferri family of de novo designed di-iron carboxylate proteins in which two iron atoms are coordinated by a combination of histidine and carboxylate residues within a self-assembling four-helix bundle. DFsc is well-folded, thermodynamically stable, and catalytically active. Recently, the chemical reactivity of this protein was altered from phenol oxidation to N-hydroxylation by the addition of a single active site histidine residue and three supporting mutations. Building on this prior work, we aim to (1) create new model proteins with additional active site carboxylate residues to explore the H2O2 vs. O2 preference in rubrerythrins, (2) create new model proteins with increased His/carboxylate ratios in the active site to determine the electronic and functional consequences of increased charge, and (3) investigate the influence of the His/carboxylate ratio on the metal-binding preferences of our de novo protein models. At the conclusion of these studies, we will have gained molecular-level insight into the structure-based factors that control biological oxidation. Our results will provide a foundation for the future development of new and improved biomimetic catalysts and protein-based therapeutic agents.

Public Health Relevance

Metal ions play key roles in the structure and function of nearly half of all known proteins, yet we have only a basic understanding of how nature optimizes the metal-protein interactions to carry out a particular chemical transformation. As disease states often arise when these carefully controlled interactions are disrupted, it is essential that we understand the connections between sequence, structure, and function in metalloproteins. The creation and characterization of small model proteins of several iron-containing enzymes will allow us to explore these connections in a controlled and systematic manner, and could eventually lead to the development of new protein-based therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM110657-01
Application #
8689205
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2014-09-05
Project End
2017-08-31
Budget Start
2014-09-05
Budget End
2017-08-31
Support Year
1
Fiscal Year
2014
Total Cost
$221,844
Indirect Cost
$21,844
Name
Ursinus College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
069889996
City
Collegeville
State
PA
Country
United States
Zip Code
19426