De Novo Designed Metallopeptides to Investigate Metal Ion Homeostasis, Electron Transfer, and Redox Catalysis.

Abstract

Protein design is a powerful way to interrogate the basic requirements for function of metal sites by systematically incorporating elements important for function. Single-stranded three-helix bundles with either thiolate-rich sites for spectroscopic characterization and electron transfer, or histidine-rich sites for redox catalysis are described. Using a previous design, two constructs were designed to incorporate a fourth cysteine residue to investigate thiolate-rich sites involved in metal ion homeostasis and electron transfer. Rational re-design replaced a putative coordinating histidine with a cysteine. A second construct embedded a CXXC binding motif into the helical scaffold. These two constructs show different UV-visisble, 113Cd NMR, and 111mCd PAC, which indicate that they form different proportions of CdS3O and CdS4. The spectroscopy of these sites sheds light on how Cd(II) bindis to CadC and suggests a dynamic site in fast exchange with the solvent. Previous attempts at the design of a rubredoxin site have focused on reproducing the peptide fold around or using flexible loop regions to define the site in addition to canonical CXXC motifs. However, the use of CXXC motifs embedded in an α-helical scaffold produces a rubredoxin site that reproduces the Mössbauer, MCD, and EPR of rubredoxin without the use of loop regions. This successful design is the largest deviation from consensus rubredoxin and zinc finger folds reported. Electron transfer rates through a de novo designed scaffold were studied by the design and synthesis of a ruthenium trisbipyridine derivative appended to an exterior cysteine residues. A redox-active tyrosine in the 70th position is implicated as a relay amino acid from the iron center and absence of the tyrosine decreases the rate of electron transfer from the metal site. This is the first photo-generated tyrosine radical in a designed protein. A construct, which was previously reported for CO2 hydration, is substituted with copper and its spectroscopic and nitrite reductase activity are studied. This is the first demonstration of nitrite reductase activity in a single-stranded designed peptide. This thesis provides insight into designed proteins and their applications and lays the groundwork for further studies to progress towards a unified multifunctional redox protein.