Design and Characterization of De Novo Photoactivated Charge Separating Proteins

Author

Paul M. Molinaro, The Graduate Center, City University of New YorkFollow

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Physics

Advisor

Ronald Koder

Committee Members

Marilyn Gunner

Matt Sfeir

Seogjoo Jang

Vikas Nanda

Subject Categories

Biological and Chemical Physics

Keywords

Protein Design, Electron Transfer, Photosynthesis, Synthetic Biology, Four Helix bundle, Charge Seperation

Abstract

Light activate charge separation can provide high energy electrons for a variety of chemical processes. Reaction center proteins serve as a scaffold for small molecules that serve as “hopping” sites for electrons. Previous reaction center designs are four helix bundles that bind at minimum two metalloporphyrin’s: a zinc porphyrin serves as a light activated electron donor, mimicking chlorophyl, and a heme molecule serving as the acceptor. The efficiency of these designs is hampered by the enhanced relaxation of the singlet excited state of zinc porphyrin molecules due to spin-orbit coupling with the paramagnetic heme acceptor. The optimal design would contain a diamagnetic “bridge” molecule between the donor and acceptor. We have recently reported improvements in our design software protCAD that make it possible to design four helix bundles that bind small molecules without strongly ligating residues. We aim to use these design tools to design a reaction center that binds two porphyrin molecules as well as hexamethylviologen. Our approach utilizes both physical tools (protCAD) and machine learning techniques (Alphafold and proteinMPNN) to design and screen charge separation scaffolds. Our first generation of designs contained only the porphyrin sites. We expressed and purified the twelve sequences that performed best in silico. Six express with heme bound, and of the six, four bind the desired zinc porphyrin. One of these designs was selected as the basis for the next generation of designs, containing a hexamethylviologen site. The design and characterization of these proteins will be discussed.

Recommended Citation

Molinaro, Paul M., "Design and Characterization of De Novo Photoactivated Charge Separating Proteins" (2024). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/6025