Peptidyl Linkers for Protein Heterodimerization Catalyzed by Microbial Transglutaminase

• Published in: Bioconjugate chemistry 2004-05, Vol.15 (3), p.491-497
• Main Authors: Tanaka, Tsutomu, Kamiya, Noriho, Nagamune, Teruyuki
• Format: Article
• Language: English
• Subjects: Catalysis; Catalysts; Cross-Linking Reagents - chemistry; Dimerization; Fluorescence Resonance Energy Transfer - methods; Kinetics; Lysine - metabolism; Microbiology; Models, Molecular; Peptide Fragments - chemistry; Peptides; Peptides - chemistry; Proteins; Proteins - chemistry; Proteins - metabolism; Ribonucleases - chemistry; Streptomycetaceae - enzymology; Transglutaminases - chemistry
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc034209o

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.