title: Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction.
abstract: Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of  Escherichia coli  alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.
authors: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Japan.
authors: Kosuke
authors: K
authors: Minamihata
authors: Masahiro
authors: M
authors: Goto
authors: Noriho
authors: N
authors: Kamiya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D043264:Phosphodiesterase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D005609:Free Radicals; D011506:Proteins; D014443:Tyrosine; D019809:Streptavidin; D006735:Horseradish Peroxidase; D043264:Phosphodiesterase I