Molecular Design And Self-Assembly Of Artificial GPx

The formation of protein functional materials in natural biological systems occurs through self-assembly of protein building blocks whose structures are encoded by organism's DNA. After transcription, translation and folding into functional building blocks in cells, the resultant individual protein components self-assemble at different size scales to form unique protein supramolecular structures with special physiological functions through molecular recognization, structural complementary and non-covalent interaction of fucntional protein building blocks in organisms. The dynamic biological assembling process of many natural protein superstructural materials involves a dynamic reversible change with physical and chemical conditions such as pH, temperature, and redox alterations.The knowledge of the details of the protein molecular recognition and supramolecular self-assembly mechanism in vivo enables us to mimic these processes in vitro through protein building block structural modification and functional redesign. The rational design of related supramolecular assemblies utilizing chemical compatibility and structural complementarity of individual protein modules based on the remarkable advance in protein engineering and biological supramolecular chemistry techniques should shed light on the principles for the development of‘bottom-up'nanotechnology and the design of functional protein nanomaterials with unique superstructures. In addition, it represents an important frontier of bionanotechnology with great potential for considerable contributions to biology, chemistry, as well as for the de novo design of biological materials with unique structures and functions.Based on the redesign of the active site and the molecular recognition mechanism of the supramolecular protein enzyme building blocks for the rational construction of potential functional biological nanomaterials, we redesigned the active site of human Grx1 and DHAR by incorporating catalytic amino acid residue Sec respectively to mimic the natural GPx catalytic function in vitro. The wild type human Grx1 and DHAR share a common substrate GSH binding site with natural GPx. In addition, we designed molecular recognition ligand of His-tag and introduced it to a C2 symmetric enzyme GST homodimer to prepare the bivalent supramolecular building block SjGST-6His, which could self-assemble to form linear enzyme nanomaterials with catalytic function through metal coordination between two different His-tags from two individual protein building blocks. At last, we discussed the principles for the redesign of the protein supramolecular building blocks in order to prepare functional biological nanomaterials and it should shed light on the the principles for the development of‘bottom-up'nanotechnology in the application of advanced functional protein nanomaterials.1. Seleno-hGrx1 with redesigned catalytic functionThe active center of human glutaredoxin (hGrx1) with excellent specific affinity for the substrate glutathione (GSH) was redesigned to introduce the catalytic selenocysteine residue for imitating the function of antioxidant selenoenzyme glutathione peroxidase (GPx) in vivo. The GSH-binding affinity of hGrx1 from human beings which shares a common thioredoxin fold with natural GPx makes it a competent candidate as antioxidant for potential medical application compared to other animal-originated protein scaffolds. Given that presence of two consecutive AGG-AGG rare codons for encoding Arg26-Arg27 residues in the reading frame of hGrx1 mRNA reduced Seleno-hGrx1 expression level significantly in the Cys auxotrophic E. coli strain BL21cysE51, a novel strategy for optimizing the rare codons was first reported and resulted in an remarkable increase of the expression level in the Cys auxotrophic cells for potential future medical production. The engineered artificial selenoenzyme displayed high GPx catalytic activity rivaling that of some natural GPx. The engineered Seleno-hGrx1 was characterized by kinetic analyses. It showed a typical ping-pong kinetic mechanism, and its catalytic properties were similar to those of some naturally occurring GPx.2. Seleno-DHAR with redesigned GPx functionDivergent evolution has resulted in superfamilies of enzymes with the same protein fold, but different catalytic performances. The disparate catalytic performances require different mechanistic steps, but some of these steps may be shared. The involved changing catalytic activity of an enzyme may just require a unique substitution of an amino acid residue at the active site. DHAR and GPx have distinct catalytic abilities, but share a common substrate GSH binding site and the same Trx fold like GST. DHAR was redesigned to incorporate catalytic Sec residue of GPx at the active site of DHAR in order to mimic the natural GPx catalytic ability by genetic engineering technique. The prepared selenium enzyme Seleno-DHAR displayed a remarkable GPx catalytic activity compared to wild type DHAR for GPx catalytic activity. And the strategy for converting DHAR catalytic activity to GPx catalytic activity is first reported and may shed light on the catalytic mechanism of natural GPx and the principles for the redesign of the function of enzyme supramolecular building blocks for rational design of the desired functional protein nanomaterials.3. Supramolecular self-assembly with catalytic functionA simple and versatile strategy for preparing functional enzyme nanowires based on self-assembly of a C2-symmetric homodimeric enzyme is proposed and demonstrated. The enzyme-loaded nanowires are fabricated throughout the outstretched His-tag recruiting a second building block by interprotein metal coordination. The prepared enzyme nanowires are characterized by tapping mode atom force microscopy. The height of one-dimensional nanowires forming at low protein concentration is about 4.0~5.0 nm, while nanowire height at the site of the entanglement crosslink of nanowires forming at higher protein concentration is about 7.0~9.0 nm, nearly twice the height of the single protein molecule (ca. 3.0~5.0 nm). The equilibrium of enzyme nanowires with individual protein blocks could be shifted and the geometric structure of the enzyme nanowires could be adjusted by modulating the concentration of the building blocks and metal ions. Given that the prepared enzyme nanowires introduce a minor difference in enzyme activity and second structure compared to individual building blocks, it is assumed that the metal-His tag coordination could enhance the enzyme nanowires thermostatic stability. The strategy may open a new avenue for designing large symmetrical materials in artificial control for a wide variety of proteins by adding His-tag to protein building blocks and shed light on the principles for the development of‘bottom-up'nanotechnology.