title: Continuous monitoring of enzymatic reactions on surfaces by real-time flow cytometry: sortase a catalyzed protein immobilization as a case study.
abstract: Only a few techniques, such as quartz crystal microbalance and surface plasmon resonance spectroscopy, enable the analysis of dynamic processes on solid supports. Here we have developed a straightforward assay based on flow cytometry to continuously follow enzymatic reactions directly on microparticle surfaces. We applied this real-time flow cytometry (RT-FCM) approach to study the covalent immobilization of green-fluorescent protein (GFPuv) on triglycine-modified polystyrene microbeads by the transpeptidase sortase A (SrtA) from Staphylococcus aureus. Though commonly treated as functionally identical catalysts, the SrtA variants SrtAΔ₅₉ and SrtAΔ₂₅, in which the N-terminal amino acid residues 1-59 and 1-25 of the native enzyme are truncated, were shown to perform very differently with regard to this particular immobilization reaction. While SrtAΔ₅₉ efficiently catalyzed the covalent attachment of GFPuv to the surface (as indicated by a linear increase of microbead fluorescence), SrtAΔ₂₅ was essentially inactive. Besides the length of the N-terminal amino acid extension on the SrtA construct, the position of the hexahistidine tag at either the N- or C-terminus affected the efficiency of enzymatic protein immobilization. Apart from three enzyme variants containing the native core structure of SrtA, we also included three recently evolved mutants of SrtA in this comparative study. With these mutants we observed a rapid initial attachment of the GFPuv target protein to the microbeads. However, with proceeding reaction time, cleavage of the covalently immobilized target protein from the surface prevailed over the coupling reaction, consequently causing a decline of microbead fluorescence. In general, the RT-FCM approach used herein represents a powerful analytical tool for qualitative dynamic studies of many heterogeneous enzymatic reactions or other binding events that influence the fluorescence properties of microparticle surfaces.
authors: Laboratory for Bioactive Materials, Empa, Swiss Federal Laboratories for Materials Science and Technology , Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland.
authors: Tobias
authors: T
authors: Heck
authors: Phu-Huy
authors: PH
authors: Pham
authors: Frederik
authors: F
authors: Hammes
authors: Linda
authors: L
authors: Thöny-Meyer
authors: Michael
authors: M
authors: Richter
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011137:Polystyrenes / Q000737:chemistry; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology; D013499:Surface Properties; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D055767:Immobilized Proteins; D011137:Polystyrenes; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases