Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase.

Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a kcat/KM value of 19 ± 3 µM-1 min-1. This sequence was then genetically appended onto a test protein as a reactive 'Q-tag' and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.

Site-specific protein labelling and immobilization mediated by microbial transglutaminase.

Microbial transglutaminase (mTG) shows broad substrate specificity that is amenable to in vitro bio-conjugation applications. Herein, test proteins were genetically fused with peptide tags, followed by mTG-mediated propargylation of their reactive Gln residues. The propargylated proteins were subjected to copper-assisted azide-alkyne cycloaddition to demonstrate either fluorescent labelling or immobilization.

Continuous enzyme-coupled assay for microbial transglutaminase activity.

Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-ε-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG's promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K(M)=3 mM) than the commonly used Cbz-Gln-Gly (K(M)=58 mM), attesting to its potential for application in biocatalysis and bioconjugation.

Recovery of red fluorescent protein chromophore maturation deficiency through rational design.

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore's covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.