title: Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase.
abstract: Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (k(cat)), rather than substrate binding affinity (K(m)). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry*; D013306:Streptomycetaceae / Q000201:enzymology; D011503:Transglutaminases / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; C023560:ribonuclease S-peptide; D011503:Transglutaminases; D012260:Ribonucleases; D008239:Lysine