Artificial Metabolons:  Design of Self-Assembled Bio-Complexes
Creators: Garcia, Kristen E.
Publisher: name: Columbia University
Year: 2017
Subjects: subject: Chemical engineering
subject: FOS: Chemical engineering
subject: Proteins
subject: Biochemistry

description: Protein-protein interactions are vital to every living organism, and it is thought that most, if not all proteins interact in some way with other proteins for purposes including for cellular metabolism, signal transduction and DNA replication. These protein complexes can range in stability from permanent to transient, and they are driven by interactions at the protein-protein interfaces including hydrophobicity, hydrogen bonding, electrostatic interactions, van der Waals interactions and covalent disulfide bonding. Many complexes, such as transient complexes of sequential enzymes called metabolons, are poorly understood. In recent years, there have been many efforts to mimic nature and engineer new protein complexes with defined spatial arrangements with increased stability and more efficient transport of the enzymatic reaction intermediates. There is much to be understood in these complexes, including the role of substrate channeling. In this dissertation, we study a natural metabolon and engineer new protein complexes.
      In our first study, we construct designed protein aggregates of the single enzyme small laccase (SLAC). SLAC is a multi-copper oxidase that can be easily genetically modified and is used as an oxygen-reduction catalyst on enzymatic bio-cathodes. A new dimeric interface is introduced, which, in combination with the threefold symmetry of the naturally trimeric SLAC, drives the self assembly of SLAC with two disulfide bonds in an oxidative environment. These enzymatically active aggregates form upon the addition of cupric ions to the purified protein, and electron microscopy shows the symmetry of the aggregates to be consistent with the design. We demonstrate improvements over the non-complexed enzyme including an increased resistance to permanent thermal denaturation and a lower reaction overpotential and increased current density when employed on an oxygen-reduction bio-cathode with single-walled carbon nanotubes incorporated into the enzyme aggregates. 
      In our next line of work, we study a natural tricarboxylic acid (TCA) cycle metabolon, focusing on two enzymes: mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS). These enzymes have long been proposed to form a spatially organized complex that facilitates substrate channeling, a process in which a reaction intermediate is transferred directly from one enzyme active site to the next without first diffusing into the bulk through mechanisms such as electrostatic interactions. Structural evidence has been difficult to obtain due to the transient nature of many of these complexes. In Chapter 3, we examine the in vitro complex structure of the recombinant enzymes and find that it is similar to the recently proposed in vivo complex structure. Furthermore, there is evidence of a positively charged electrostatic channel connecting the enzyme active sites along which the oppositely charged reaction intermediate can travel by bounded diffusion. Site-directed mutagenesis along the channel on CS results in inhibited substrate channeling. 
      Finally, we develop a platform to study substrate channeling in engineered multi-enzyme complexes. Efforts to engineer multi-enzyme complexes in recent years have made use of protein and nucleic acid-based scaffolds. Many of these complexes exhibit increased coupled enzymatic activities, but there is a question of what effects are due to substrate channeling and how to apply these strategies to any enzyme pair. In this work, we attach CS and the non-channeling cytosolic malate dehydrogenase to DNA and engineered protein cage scaffolds. These assemblies retain their enzymatic activities, and these methods can be used to study substrate channeling in many enzyme pairs including the naturally channeling and inhibited channeling TCA cycle enzymes.