Novel enzymatic activity derived from the Semliki Forest virus capsid protein.

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k(cat)/K(M) values up to 5 x 10(5) s(-1) M(-1). The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK(a1)=6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k(2)=4.4+/-0.6 s(-1) and k(3)=1.6+/-0.5 s(-1), respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.

Combined affinity and catalytic biosensor: in situ enzymatic activity monitoring of surface-bound enzymes.

Surface Plasmon Resonance Spectroscopy (SPR) and miniature Fiber Optic Absorbance Spectroscopy (FOAS) were combined to monitor in situ and quantitatively an enzymatic model reaction catalyzed by beta-lactamase. The enzyme was covalently immobilized to the gold surface of a SPR chip, which was functionalized with NeutrAvidin through a biotinylated alkanethiol self-assembled monolayer, thus serving as a highly sensitive affinity biosensor. SPR was used to control the density of the surface-bound enzyme. Nitrocefin as the enzymatic substrate was allowed to react with the immobilized enzyme in the SPR flow cell, and its turnover was detected with the FOAS system acting as the catalytic biosensor. The coupling of the two techniques has a substantial potential for highly controlled on-line monitoring of surface-bound enzyme activity. The FOAS technique may also be easily employed as an add-on device to other types of affinity sensing instruments.

NTA-Functionalized Poly(L-lysine)-g-Poly(Ethylene Glycol): A Polymeric Interface for Binding and Studying 6 His-tagged Proteins.

In this paper, a novel graft copolymer, poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) with part of the PEG chains carrying a terminal nitrilotriacetic acid group (NTA) was synthesized. Through electrostatic interactions, these polycationic graft co-polymers assemble spontaneously from aqueous solution onto negatively charged surfaces, forming polymeric monolayers that present NTA groups at controlled surface densities on a highly PEGylated background. The NTA-functionalized PLL-g-PEG surfaces proved to be highly resistant to nonspecific adsorption in contact with human serum while allowing the specific and reversible surface binding of GFPuv-6His and ß-lactamase-6His in native conformation. Micropatterns consisting of NTA-functionalized PLL-g-PEG in a background of PLL-g-PEG were produced using the "molecular assembly patterning by lift-off" technique. Exposure to Ni2+and GFPuv-6His resulted in a protein pattern of excellent contrast as judged by fluorescence microscopy. Furthermore, optical waveguide lightmode spectroscopy (OWLS) and a miniature fiber optic absorbance spectrometer (FOAS) were combined as affinity and catalytic biosensor to monitor in situ and quantitatively the amount of immobilized ß-lactamase-6His and to determine the activity of the immobilized enzyme. The NTA-functionalized PLL-g-PEG surface is considered to be a promising sensor platform for binding 6 His-tagged proteins thanks to the simplicity and cost-effectiveness of the surface modification protocol, high specificity and nearly quantitative reversibility of the protein binding, and the potential to fabricate microarrays of multiple capture molecules.

Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing.

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.