Engineering of FhuA into an ideal scaffold for metallozymes

Metallozymes are composed of a biomolecular scaffold harboring a metal complex. Such hybrid systems connect the “best” features of enzymatic (biomolecule) and homogenous (metal) catalysis. Chemogenetic optimization of metallozymes can lead to a wide range of applications in biotechnology and medicine. In this perspective, catalysis with a focus on broad reaction scopes, high activity and selectivity under mild reaction conditions as well as the development of artificial restriction enzymes for gene correction are the current topics of interest. The use of membrane proteins as proteinshell for metallozymes could broaden the range of applications due to their rigid and defined structure, structural integrity in e.g. organic solvents and at elevated temperatures as well as their easy modifiability. Still, researchers forgo the area of membrane proteins because the handling of the protein is highly challenging and requires lots of skillfulness. The constant presence of detergents mimicking the native protein environment is a drawback just as the expression and extraction in large scale with “good” quality. Crystal structures are rarely available and to set-up analytics is not easy. This thesis overcame the challenges of the development of a metallozyme based on a transmembrane β-barrel protein (FhuA) of E. coli. FhuA, made of 22 β-strands, was engineered and expression/extraction was optimized to make it a suitable scaffold. To gain catalytic activity, a ruthenium based cyclopentadienyl metal complex [Ru-Cp*] was coupled to the engineered FhuA. Analytical methods for FhuA metallozyme characterization were developed. In the first chapter, different FhuA variants were (re)engineered and molecular dynamic computer simulations were used to analyze the surrounding of the catalyst binding site. Following, for the developed variants, expression conditions in terms of media, temperature and time were optimized. Expression was scaled-up from 0.25 L flask expression to 10 L fermentation. Extraction was compared by protein solubilization with different detergents and harsh membrane dissolution with subsequent protein precipitation using organic solvents. The protein could be refolded by the polymer polyethylene-polyethylene glycol (PE-PEG). Regarding good yields and purity, protein solubilization with dodecylmaltoside followed by n-hexane-EtOH-SDS dissolution proved to be superior to other detergent and organic solvent based extraction methods, achieving up to 800 mg protein with a purity of 90 % from one liter extracted culture broth. Stability investigations in the organic cosolvents tetrahydrofuran (THF), ethanol (EtOH), chloroform/methanol (C/M) and dioxane were performed in the second chapter. Highest stability of selected variants was obtained in up to 40 vol% THF, 25 vol% dioxane, 10 vol% EtOH or C/M. Basic pH values are preferable over acidic ones and the variant FhuA Δ1-159_C556 (having least mutations referring to the wildtype DNA sequence) showed higher temperature stability than FhuA Δ1-159_C556_tev, possessing more mutation (melting point 74 °C vs. 55 °C). The third chapter focused on the development and characterization of the metallozyme based on FhuA-[Ru-Cp*]. The coupling efficiency was verified by cysteine titration and analyzed by MALDI-TOF-MS. Structural integrity after coupling was confirmed by CD spectrophotometry. In summary, in this thesis, for the first time, a large, highly hydrophobic engineered membrane protein was used as a biomolecular scaffold for the development of a metallozyme. Advantages of using FhuA are its high purity and tremendous stability in organic cosolvents, elevated temperatures and basic pH values. After assembly of the protein and the metal catalyst, the system was characterized and provides fundamentals for the use of other membrane proteins in metallozyme applications. Additionally on a shortterm basis, the herein designed protein including the developed methods is used for the generation of a “platform catalytic system”. Table of context 1 ABBREVIATIONS . 1 2 INTRODUCTION . 3 2.1 State of the art: metallozymes . 3 2.2 Biomolecular scaffold and metal complex . 8 2.3 Thesis objective . 11 3 ANALYTICAL METHODS . 13 3.1 Circular dichroism (CD) . 13 3.2 Differential scanning calorimetry (DSC) . 16 3.3 Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). 17 4 EXPERIMENTAL CHAPTER A-C . 21 Chapter A ENGINEERING, EXPRESSION, EXTRACTION, PURIFICATION . 21