Genetic Incorporation of an Unnatural Fluorescent Amino Acid in a Plant H-ATPase Expressed in Yeast

Abstract

Fluorescence labeling provides an important tool to study the structure and function of proteins. Typically, fluorescence labeling involves either chemical modification or protein fusion with fluorescent probes. These techniques, however, have several significant drawbacks. First, the degree to which fluorescent probes interfere with protein folding and function is a concern. Second, many protein domains are difficult to target specifically. In the particular case of integral membrane proteins, few fluorescent probes can be targeted specifically to transmembrane domains without significantly disturbing protein function. Thus, small fluorescent probes that minimally perturb the structure and function of integral membrane proteins are needed. In this study, we genetically incorporated Anap, an environmentally-sensitive fluorescent unnatural amino acid, into specific locations in the Arabidopsis thaliana type 2 H+-ATPase (AHA2) expressed in Saccharomyces cerevisiae. The nonsense stop codon TAG was substituted for Trp codons to incorporate the unnatural amino acid into specific sites within the first two transmembrane alpha helices of AHA2 using an engineered suppressor tRNA that carries the artificial amino acid and is orthogonal to natural amino acids. Culture conditions were varied to maximize expression of a fluorescent 97 kDa protein in yeast that was only observed when Anap was included in the culture media. By taking advantage of the environmentally sensitive properties of this fluorescent unnatural amino acid, the resultant labeled protein can be used to study conformational dynamics during the enzyme cycle that may result from changes in the local environment surrounding the incorporated fluorescent probe. Fluorescence labeling of membrane proteins using this approach should allow detailed studies of local conformational kinetics that will shed new light on structure function relationships in this class of enzymes.