Title

De Novo Design of Metalloproteins with Variant Hydrophobic Cores

Date of Award

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

Michael Y. Ogawa, PhD

Second Advisor

George S. Bullerjahn, PhD (Committee Member)

Third Advisor

Thomas H. Kinstle, PhD (Committee Member)

Fourth Advisor

Moira van Staaden, PhD (Committee Member)

Abstract

This work studies a series of related de novo designed luminescent Cu(I) metalloproteins having different cysteine-based metal binding sites within the hydrophobic cores. The peptide sequences are based on the (Ia-Eb-Ac-Ld-Ee-Gf-Kg)n heptad repeat which is known to form two-stranded coiled coil in aqueous solution. The metal-binding cysteine residues are placed at the hydrophobic a and/or d positions in the middle of the peptide sequences. With subtle changes of the metal-binding site, the resultant Cu(I) metalloproteins give distinctly different structures and properties. The peptide C16C19GGY exists as a random coil and the Cu(I)-C16C19GGY metalloprotein exists as a 4-helix bundle (Cu4P4, P = peptide) which gives strong room temperature emission at 600 nm. The peptide C12C16GGY exists as a random coil and the Cu(I)-C12C16GGY metalloprotein exists as a 4-helix bundle (Cu4P4) which gives strong room temperature emission at 575 nm. The peptide C12C16C19GGY exists as a random coil and the Cu(I)-C12C16C19GGY metalloprotein exists as a 4-helix bundle (Cu8P4) which gives strong room temperature emission at 605 nm. The peptide C12C19GGY exists as a random coil and the Cu(I)-C12C19GGY metalloprotein also exists as a random coil (Cu4P4) which gives strong room temperature emission at 600 nm. The peptide C16GGY, C19GGY exist as two-stranded coiled-coils and the Cu(I)-C16GGY, Cu(I)-C19GGY metalloproteins exist as 4-helix bundles (Cu3P4) which gives very weak room temperature emission at 603 nm and 630 nm respectively.