Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

Abstract

Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F n Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F n Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F n Y at residue 356. The procedure to generate 100 mg quantities of Y 356 F n Y-R2 takes 3-4 months. INTRODUCTION Problem under investigationThe Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of all four nucleotides to their corresponding 2¢-deoxynucleotides and consists of two homodimeric subunits: R1 and R2 (refs. 1-3). R1 is the homodimeric subunit where nucleotide reduction occurs. It contains binding sites for nucleoside diphosphate substrates as well as for deoxynucleoside triphosphate and ATP allosteric effectors, which govern substrate specificity and turnover rates. R2 is the homodimeric subunit, which houses the diiron-tyrosyl radical cofactor (Y 122 ) essential for catalysis. Each turnover requires radical migration from the Y 122 site on R2 to the active site of R1, over a distance of 35 Å . The mechanism of this long-range radical propagation event is an active area of research 4,5 .Structures of active RNR, a 1:1 complex of R1 and R2, are not available. However, Uhlin and Eklund 6 have generated a docking model of this complex from the individual structures of R1 and R2 (refs. 7,8

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli

“…In the F-Tyr sample, a mass shift around 17 U was observed for the seven peptides fragments (Table 1). Our result correlated well with earlier reports on the efficient incorporation of F-Tyr into recombinant protein (Prajna et al 2005;Seyedsayamdost et al 2007;Wilkins et al 2010). …”

“…Homogeneous proteins containing fluorine atoms have been frequently synthesized through the combination of chemical peptide synthesis and expressed protein ligation technique (Wilkins et al 2010). This method is limited to certain locations within a protein, typically the C-terminus, and can be technically challenging for proteins that are sensitive to denaturation (Seyedsayamdost et al 2007). Due to its significance, the in vivo incorporation of fluoro-amino acids into recombinant proteins is essential.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…Remember, the pK a of the phenolic proton which is 10 in tyrosine varies between 5.2 and 9.0 in its fluorinated analogs depending on the level and position of fluorination. 68,70 Thus, the fluorinated Tyr analogs can be used in order to investigate acid-base catalysis or redox potential properties. In addition, fluorinated tyrosine radicals are characterized by peak reduction potentials between 705 and 968 mV whereas Tyr as a reduction potential of 642 mV.…”

Inteins in Protein Engineering

“…This allows the EPL precursor to be immobilized and purifi ed on a chitin column, where the target protein segment is joined to the intein by a stable α -thioester. The column is then washed with a thiol -containing small molecule, such as thiophenol [32] or 2 -mercaptoethanesulfonic acid ( MESNA ) [33] , and the desired synthetic peptide with an N -terminal Cys is added. The small thiol -containing molecule creates a more reactive α -thioester, which is more easily displaced by the semi -synthetic peptide to create the ligated product protein.…”

“…Because EPL allows an expressed protein to be joined to a synthetic peptide, it permits almost any protein to be post -translationally modifi ed with a wide variety of compounds. These can include biophysical probes, such as fl uorescent tags [33] or spin resonance probes [34] , or it can be used to generate proteins that incorporate unnatural amino acids [44] or other prosthetic groups. For example, EPL has been used for controlled hyperphosphorylation in vitro to study specifi c protein -protein interactions in the transforming growth factor -β ( TGF β ) receptor pathway [45,46] .…”