Isolating, characterizing, and engineering novel Cu-proteins and peroxidases

Metalloproteins are a fascinating class of proteins that function at the heart of several important biological processes including photosynthesis, respiration, and nitrogen fixation. It is even more amazing, considering that nature uses a small set of tertiary structures and metal centers to perform all these different functions with efficiency and selectivity. How nature tunes the activity within these scaffolds has been the area of research for many years. The goal of this work is to understand the underlying mechanisms of such tuning with a special focus on the role of subtle changes of residues in the secondary coordination sphere of the metal ion, an underexplored area of study. I use protein engineering techniques not only to shed light on the mechanisms underlying such changes, but also to design new functionalities within our scaffold proteins and to enhance their properties for specific purposes, such as fuel generation. This work is divided into three main sections. In the first, I focus on characterizing a novel metalloprotein, N. mar_1307, from the organism Nitrosopumilus maritimus. While the protein shares a protein fold and Type 1 copper coordination site with other common electron transfer cupredoxins, the lack of an axial residue creates an open binding position in the Cu center, leading to a novel enzymatic function, NO oxidation. The purification, characterization, and activity assays of the protein are described in detail in chapter 2. The second and major focus of this work is on tuning the reduction potential of azurin, a common electron transfer protein. In chapter 3 I demonstrate that how by making mutations around the Cu site, and replacing Cu with Ni I can obtain an azurin variant with a reduction potential of nearly 1V, the highest potential that can be observed under physiological conditions, along with other variants with negative potentials. Chapter 4 describes the characterization of a series of Phe114 mutants that were used to understand the role of this critical secondary sphere residue in tuning the reduction potential of the Cu site. Chapter 5 demonstrates the Marcus inverted region of electron transfer in a series of azurin variants with different reduction potentials. Finally, I show my initial attempts toward the design of a high-throughput screening platform for the directed evolution of azurin in chapter 6. In chapters 7 and 8, I focus on the design of novel functionalities in one of our model scaffolds, cytochrome c peroxidase (CcP). Chapter 7 describes the work done to enhance the Mn(II) oxidation activity in a designed model of manganese peroxidase within the CcP scaffold based on modifications of the second coordination sphere around the Mn(II) binding site. In chapter 8 I report the design and characterization of a novel CcP variant that shows catalase-like activity in “as-purified” form and forms a heme-protein crosslink in the heme-bound form.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2016

Author dc:creator

• Hosseinzadeh, Parisa

Contributors dc:contributor

• Lu, Yi
• Gennis, Robert R
• Tajkhorshid, Emad
• Zhao, Humin

• Protein design
• Redox Potential tuning
• Secondary coordination sphere
• metalloprotein
• azurin
• cytochrome c peroxidase
• N-mar 1307

Statement dc:rights

• Copyright 2015 Parisa Hosseinzade

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/89172

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/89172