Background

The covalent modification of proteins to investigate their biochemical properties (Means and Feeney, 1971) was initially limited by the often uncertain location of amino acid side chains in protein tertiary structure, and the variable reactivity of different side chains with extrinsic chemical probes. However, the combination of protein sequence information, crystallographic depictions of protein folding, site-directed mutagenesis, and specific alkylation chemistry allowed high yield, often stoichiometric labeling of genetically engineered cysteine (Cys) sulfhydryls at strategic sites in proteins. Cys sulfhydryls are selectively modified by reagents like methanthiosulfonates, maleimides, and iodoacetamides (Means and Feeney, 1971). When combined with site-directed spin labeling experiments and electron paramagnetic resonance (EPR) spectroscopy, this approach provided information about the structure and function of membrane proteins (Altenbach et al., 1989, 1990). In addition, while exploring the properties of the major facilitator lactose permease, LacY, Kaback and colleagues adapted the method to site-directed fluorescent labeling (Nie et al., 2007; Smirnova et al., 2008). However, LacY is a Gram-negative bacterial inner membrane (IM) protein that is shielded by the barrier properties of the outer membrane [OM; Nikaido, 2003; Nikaido and Vaara, 1985] and hence recalcitrant to labeling in vivo. Our protocol focuses on fluorescent modification of engineered Cys residues on the external loops of Gram-negative bacterial OM proteins (OMPs), to transform them into sensors. This methodology enables labeling of living cells, which has many advantages, including facile preparation, high sensitivity, biochemical durability, multifunctionality, and convenient storage (Nairn et al., 2017; Chakravorty et al., 2019; Kumar et al., 2022). We also genetically engineered, purified, and fluorescently labeled binding proteins [e.g., human siderocalin (HsaSCN)] for use as soluble sensors. These manipulations transform both types of proteins into sensitive, quantitative biosensors that detect the presence of biochemicals, metabolites, natural products, enzymes, and other molecules.