posted on 2019-02-06, 19:10authored byStevie Norcross, Ashwin Sunderraj, Mathew Tantama
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.