Engineering the interface of cytochrome b5 and myoglobin for in vitro and in vivo applications

Heme-copper oxidases (HCOs) are large membrane proteins found in both bacteria and in eukaryotes. They catalyze about 90% of oxygen reduction in the atmosphere. They utilize a heme-copper center, which is comprised of a heme and a copper ion coordinated with three histidines, to catalyze the four-electron reduction of oxygen to water. Unfortunately, native enzymes are difficult to study because they are difficult to purify, and the presence of multiple metal cofactors make it difficult to characterize. Therefore, there is a need for small protein models that are able to mimic the native systems. Our lab was able to incorporate the heme-copper center found in HCOs in a smaller scaffold, Myoglobin, and the resulting mutant was named CuBMb. When our protein model was paired with its physiological electron transfer partner, cytochrome b5, fast electron transfer and O2 consumption was achieved, making our protein model system to have a catalytic rate similar to that of native HCOs. A key to the above success was engineering the electrostatic interactions between CuBMb and cytochrome b5. As a result, the system works well only when the ionic strength of the buffer is <5 mM, which is outside the common physiological ionic strength. As a result, applications for this system are limited. To overcome this limitation, cytochrome b5 was engineered such that the interaction with myoglobin became hydrophobic, which allowed for a broader application scope. The mutants were designed using Rosetta, and their oxidase activity was characterized. However, after two rounds of Rosetta, the designed mutants had lower oxidase activity compared to the native at any ionic strength.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2018

Author dc:creator

• Chan, Tracy Ying

Contributors dc:contributor

• Lu, Yi

• metalloproteins
• protein-protein interfaces

Statement dc:rights

• Copyright 2018 Tracy Chan

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/101364

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/101364