Metalloprotein engineering with unnatural amino acids: application in functional heme-copper oxidase and azurin

Yang, Yu

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https://hdl.handle.net/2142/49778 Copy

Description

Title

Metalloprotein engineering with unnatural amino acids: application in functional heme-copper oxidase and azurin

Author(s)

Yang, Yu

Issue Date

2014-05-30T17:17:14Z

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Gennis, Robert B.
• Gruebele, Martin
• Luthey-Schulten, Zaida A.

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Computnl Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Heme-copper oxidase
• azurin
• unnatural amino acid
• protein engineering
• electron paramagnetic spectroscopy

Abstract

Metalloproteins, proteins with metal ion cofactors, are estimated to make up more than a third of total proteome. They involve in key biological processes such as photosynthesis, respiration, and nitrogen fixation. The important roles and potential applications of metalloprotein urges the need for metalloprotein engineering. The advent of modern molecular biology provides us the ability to mutate certain amino acid in metalloproteins to all other natural amino acids through site-directed mutagenesis. However, site-directed mutagenesis is restricted to 20 natural amino acids, only half of which could coordinate to metal. Incorporation of unnatural amino acids greatly expands the toolbox of bioinorganic chemists. Several methods for unnatural amino acid incorporation have been developed, including cavity complementation, total synthesis, native chemical ligation, expressed protein ligation and amber suppression. They all have their own advantages and disadvantages. Heme copper oxidases (HCOs) catalyzes reduction of O2 to H2O and harvest energy at the end of respiration chain. The enzyme plays a pivotal role in aerobic respiration and thus is important for life on earth. The oxygen reduction reaction is catalyzed in a dinuclear center which is composed of a heme and a copper ion coordinated with three histidines. Besides that, there is a conserved tyrosine residue which forms a post-translational modified His-Tyr cross-link with one of the three histidines at CuB site. Due to the fact that HCO is a large membrane protein with multiple metal cofactors, it is difficult to study and there are many unsolved questions. In particular, since His-Tyr cross-link forms simultaneously in vivo, it has been difficult to directly probe the effect of cross-link. Based on a model protein system developed in our lab, we have been able to use amber suppression method to incorporate an unnatural amino acid that mimics cross-linked His-Tyr. The new protein, termed imiTyr-CuBMb, is able to perform O2 reduction reaction three times as fast as protein without cross-link and in a much cleaner fashion. Tyrosine is a conserved redox-active amino acid that plays important roles in heme-copper oxidases. Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O2 reduction condition remains elusive. Using a functional oxidase model in myoglobin called F33Y-CuBMb, we observed of radical under H2O2 reaction condition by electron paramagnetic resonance (EPR) spectroscopy. Simulation of X- and Q-band EPR spectra suggested this radical is a tyrosyl radical, while natural amino acid mutagenesis and unnatural amino acid incorporation proved this radical is at Tyr33, the active site Tyr in F33Y-CuBMb. Freeze quench technique was used to trap the same radical under O2 reduction condition, confirming the active site Tyr works as electron and proton donor in the reaction. Furthermore, through incorporation of a series of Tyr analogues, effect of pKa and redox potential of Tyr on enzymatic activity was explored. Native HCOs have efficient electron transfer pathway toward binuclear center. In order to increase the electron transfer rate toward our protein model, the physiological electron transfer partner, cyt b5 was used as electron donor. Mutations were introduced to Mb to increase interaction between engineered Mb and cyt b5. The resulting system has faster electron transfer and significantly improve O2 consumption rate. Azurin is a Type 1 copper protein involved in biological electron transfer. The copper ion in azurin is coordinated by two histidines and one cysteine in equatorial position and methionine, backbone oxygen of glycine at axial position. The methionine and cysteine have been replaced by unnatural amino acids using expressed protein ligation. His117 is believed to play important role in electron transfer and redox potential tuning. This amino acid was replaced with a series of His analogues to explore the effect of pKa of His in redox potential. Azurin, as well as other Type 1 copper proteins, has Greek key β barrel scaffold. Circular permutation, a method that changes amino acid sequence of a protein with little perturbation to its three dimensional structure, was carried out in azurin. The resulting mutants were characterized structurally and spectroscopically.

Graduation Semester

2014-05

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http://hdl.handle.net/2142/49778

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Copyright 2014 Yang Yu

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