Surface-tethered protein switchesw

Biological sensor platforms usually combine a recognition element (e.g. antibody, recombinant protein/peptide, or oligonucleotide/oligonucleoside) that binds a target molecule (e.g. protein, DNA fragment, or whole cell) with a transduction/ output scheme that provides a readable signal. A class of proteins called protein switches represent a unique platform for biosensors since they combine both input and output domains. Protein switches are engineered fusion proteins with two domains: an input domain that recognizes and responds to an input signal and an output domain whose function is regulated by the state of the input domain. This feature can be exploited to selectively switch the activity of a protein on and off through recognition and binding of the input substrate. For example, the fusion of a ligand-binding domain with an enzyme domain can result in a protein switch in which enzyme activity (the output function) is regulated by ligand concentration (the input signal) via ligand-induced conformational changes. Such modular allosteric regulation allows the possibility of developing universal biosensors in which different input domains can be coupled to the same output domain. The response of a protein switch to an input signal is usually measured in solution, however, for sensor applications it is desirable to immobilize the recognition element. Here we demonstrate a fully functional surface tethered protein switch, the first step towards a platform for universal biosensors. For this purpose we used RG13, an engineered allosteric protein switch comprising a maltose binding protein domain (MBP) and TEM1 b-lactamase domain (BLA), a protein that hydrolyzes b-lactam antibiotics. Scheme 1 shows a schematic illustration of the strategy for the immobilization of RG13 on the gold surface (see ESIw for details). To ensure proper orientation for ligand and substrate accessibility, RG13 was genetically engineered with a hexahistidine (His6) tag at the C-terminus, which served as the site for attachment to the surface via binding to chelated Ni(II). The switch was immobilized on a gold surface using a nitrilotriacetic acid (NTA)-terminated thiol chelated with nickel(II) (see Scheme 1). First, a mixed monolayer of OHterminated thiol (SH(CH3)11OH) and NH2-terminated thiol (SH(CH3)11NH2) was formed on ultra-smooth template stripped gold. A terminal aldehyde group was then formed on the NH2-terminated thiols (switch attachment sites) by reaction with glutaraldehyde. Subsequent reaction with N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) couples the NTA group to the thiol through formation of a schiff base with the aldehyde group. After immersing in NiSO4 solution, the functionalized surface was incubated with