Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.

Assembly of type 1 pili from Escherichia coli is mediated by FimC, a periplasmic chaperone (assembly factor) consisting of two immunoglobulin-like domains. FimC is assumed to recognize the individual pilus subunits in the periplasm mainly via their conserved C-terminal segments and to deliver the subunits to an assembly platform in the outer membrane. Here we present the first biochemical characterization of a periplasmic pilus chaperone and analyze the importance of the two chaperone domains for stability and function. Comparison of the isolated C-terminal domain with wild-type FimC revealed a strongly reduced thermodynamic stability, indicating strong interdomain interactions. The affinity of FimC toward a peptide corresponding to the 11 C-terminal residues of the type 1 pilus adhesin FimH is at least 1000-fold lower compared to binding of intact FimH, confirming that bacterial pilus chaperones, unlike other chaperones, specifically interact with folded pilus subunits.