Chemical synthesis of proteins: a tool for protein labeling

An ingenious stratagem useful to understand and modulate the structural and functional features of the proteins refers to the modification of their chemical structure. In this regard, the chemical synthesis of proteins appears a key tool, as it allows the unlimited modification of a polypeptide chain with any kind and number of labels. The last decade has seen the introduction of several techniques of chemical protein synthesis that allow the manipulation of proteins structure [Hahn, M.E. & Muir, T.W. 2005; Kent, S.B.H. 2009]. Among these approaches, the Native Chemical Ligation (NCL) appear as the most useful and advantageous strategy [Dawson, P.E. et al. 1994]. The method leads to the formation of a protein molecule through the chemoselective ligation of two peptide segments containing the former a C-terminal α-thioester group and the latter an N-terminal Cys residue. Each peptide segment is prepared by solid-phase peptide synthesis (SPPS) and thus can contain any useful chemical modifications, such as unnatural amino acids, post-translational modifications, isotopes and fluorophores. A semisynthetic version of NCL is the Expressed Protein Ligation (EPL), in which one or both of the peptide building blocks are made by recombinant DNA expression, while the actual ligation step is still a chemical reaction [Muir, T.W. et al. 1998; Evans T.C., et al. 1998]. This PhD thesis represents a practical guide to protein chemical synthesis and modification, as it presents and discusses a general protocol useful for the preparation of proteins site-specifically functionalized with two molecular probes. The designed synthetic approach is based on the use of EPL and thus requires the splitting of the protein of interest in two fragments. The N-terminal protein fragment is expressed by recombinant means and is endowed of a C-terminal thioester group. Such fragment can be selectively labeled on its unique Cys residue with a probe containing a thiol-reactive moiety. After first labeling, the thioester fragment is ligated to the C-terminal remaining protein portion, which is synthesized by SPPS and carries an N-terminal Cys residue, affording the full-length mono-labeled protein. The Cys residue involved in the ligation reaction is finally exploited to introduce the second probe into the protein, giving the doubly-functionalized full protein molecule. The effectiveness of the proposed semisynthetic protocol has been assessed through the preparation of a series of CTPR3 (Consensus Tetratrico Peptide Repeat protein 3) protein variants in which two fluorophores are incorporated at different, specific positions. The protocol enabled the preparation of four homogeneously doubly-labeled variants, defined CTPR3[N_C], CTPR3[1_C], CTPR3[2_C] and CTPR3[1_3], whose name suggest the positions in which the two fluorophores are located. The obtained fluorescent variants are useful for the conduction of folding studies by FRET (Förster resonance energy transfer). In this thesis are presented the results of a preliminary biophysical investigation performed on the obtained fluorescent CTPR3 variants by circular discroism and FRET. Such analysis opens the way to further folding studies on the fluorescent variants of CTPR3 by the use of modern biophysical spectroscopic methods, such as the single molecule approaches.