Protein engineering for structure-function studies of bioactive (macro)molecules

Abstract

The present Ph.D. project deals with the use of protein engineering techniques to express in recombinant form and characterize disulphide rich bioactive peptides, such as conotoxins. Moreover, taking advantage of their high target specificity and stability, the potential application of this class of macromolecules in “green” (environmental) and “white” (industrial) biotechnology fields have been explored. Protein engineering is a powerful tool to improve some proteins parameters, acting on their sequence and structure, or to insert a desired function. There are three basic approaches in protein engineering: the rational design is an "intelligent" design that aims to change the structure or function of a protein, starting from the analysis of its three-dimensional structure. Its nucleotide sequence can be modified and the protein finally “built” and characterized (Hellinga, 1997). The second approach is the directed evolution or DNA breeding, a technique that simulates biological evolution mechanisms (Stemmer, 1994). In fact it involves the generation of random mutations in the gene coding for a protein, or shuffling the genes encoding different domains, to generate a high number of new proteins that will be then selected for a desired function. The third one, named de novo design, allows to design and build artificial proteins which don’t exist in nature, with novel functions and properties (Dahiyat and Mayo, 1997). This strategy takes advantage of computational biology to draw novel proteins both using natural scaffolds as templates and starting completely from scratch (Dahiyat and Mayo, 1997). The idea of using stable protein structural motifs as scaffold for reproducing functional epitopes or stabilize bioactive conformations is currently one of the most successful approaches of protein engineering. The knottin family of proteins is particularly interesting from this point of view, since they share a common structural fold while having very different sequences and functions in animals and plants (Norton and Pallaghy, 1998). The multiple disulphide bridges that characterize this protein family give the greatest contribution to the structure stability, allowing a high variability of the remaining amino acid sequence regions. Among knottins, the highest tolerance to sequence variability is observed in conotoxins, small disulphide-rich neurotoxic peptides extracted from cone snails venom. Nature, during the evolution, has engineered this stable scaffold to express a great variety of neurotoxins with a remarkable high target specificity for different ion channels, both voltage-gated and ligand-gated (Olivera and Teichert, 2007). As a result, their scaffold represents an excellent starting point for design strategies aimed at developing new miniproteins with new properties as well as more stable/active conotoxins for therapeutics (Clark et al., 2005). From the natural source, the purification of conotoxins proved to be very difficult because of the large number of specimens needed to obtain a sufficient amount of venom to process. Thus, during this thesis work, a recombinant expression system has been developed to express conotoxins as GST-fusion proteins, overcoming both the problem of the small amount of a single conotoxin that can be extracted from the Conus venom, and their propensity to form insoluble aggregates because of the formation of intermolecular disulfide bridges. As a test case, the new protocol has been used to express conotoxin Vn2 from the Mediterranean Sea Conus ventricosus, a 33 amino acids long highly hydrophobic neurotoxic peptide, and its Asp2His mutant (Spiezia et al., 2012). This first part of the work involved cloning of both the conotoxins in pET-CM plasmid and their purification by affinity chromatography, exploiting the GST tag. Since the worm-hunting C. ventricosus has an array of toxins acting on invertebrate ion channels, GST-conotoxins have been tested for their neurotoxic activity on the larvae of the moth Galleria mellonella, taken as an insect model system (Spiezia et al., 2012). Further, using ω-conotoxin GVIA as template, novel metal-binding peptides were designed to generate novel biocatalysts. Through computational modeling, two disulphide bridges in the original conotoxin have been replaced with His residues and the redesigned peptides, named Cupricyclin-1 and Cupricyclin-2, have been synthesized and characterized for their copper binding ability and superoxide dismutase activity (Barba et al., 2012). The last part of the work concerned the generation of Cupricyclin-1 mutants with the aim to improve its superoxide dismutase activity and to change metal-binding specificity from copper to iron and manganese. The new expression system developed in the first part of the present project was successfully used to express these Cupricyclin mutants as GST-fusion proteins. Following GST tag removal using thrombin cleavage, the mutants have been purified and their metal binding properties analyzed by optical and fluorescence spectroscopy and mass spectrometry. Further studies will be necessary to elucidate the redox properties of these novel Cupricyclins and their potential as biosensors for the detection of heavy metals or as therapeutics in oxidative stress injuries, given the ability of Cupricyclin-1 to dismutate O2- radicals.