Abstract
Bacterial ATP-binding cassette transporters are a superfamily of transport systems involved in the import of various molecules including amino acids, ions, sugars, and peptides. In the lactic acid bacteria Lactococcus lactis, the oligopeptide-binding protein A (OppA) binds peptides for import to support nitrogen metabolism and cell growth. The OppA protein is of great interest because it can bind peptides over a broad variety of lengths and sequences; however, current methods to study peptide binding have employed low throughput, endpoint, or low dynamic range techniques. Therefore, in this study, we developed a fluorescence anisotropy-based peptide-binding assay that can be readily employed to quantify OppA function. To test the utility of our assay, we characterized the pH dependence of oligopeptide binding because L. lactis is commonly used in fermentation and often must survive in low pH environments caused by lactic acid export. We determined that OppA affinity increases as pH or temperature decreases, and circular dichroism spectroscopy further indicated that acidic conditions increase the thermal stability of the protein, increasing the unfolding transition temperature by 10 °C from pH 8 to pH 6. Thus, our fluorescence anisotropy assay provides an easy technique to measure peptide binding, and it can be used to understand molecular aspects of OppA function under stress conditions experienced during fermentation and other biotechnology applications.