pH- and Temperature-Dependent Peptide Binding to the Oligopeptide-Binding Protein A Measured with a Fluorescence Anisotropy Assay.

ACS Omega

Department of Chemistry, Institute for Integrative Neuroscience, and Institute for Inflammation, Immunology, and Infectious Disease, Purdue University, 560 Oval Drive Box 68, West Lafayette, Indiana 47907, United States.

Published: February 2019

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 , 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 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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396125PMC
http://dx.doi.org/10.1021/acsomega.8b02427DOI Listing

Publication Analysis

Top Keywords

peptide binding
12
oligopeptide-binding protein
8
fluorescence anisotropy
8
anisotropy assay
8
lactic acid
8
oppa function
8
oppa
5
ph- temperature-dependent
4
temperature-dependent peptide
4
binding
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!