Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI5-24). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His6 tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.
Bibliographical noteFunding Information:
The authors thank Dr. Dan Mullen for assistance in the production of synthetic peptides used as standards in this study, Dana Reed at the Department of Chemistry Mass Spectrometry Facility for assistance in acquiring ESI-TOF data, and Jinny Johnson at the Protein Chemistry Laboratory at Texas A&M University for assistance with amino acid analysis of synthetic peptides. This project is funded by NIH Grants GM64742 and HL080081 (G.V.) and GM08700 (L.R.M.). The University of Minnesota NMR facility is supported by NSF funding BIR-961477 and the University of Minnesota Medical Foundation.
Copyright 2009 Elsevier B.V., All rights reserved.
- Isotopic labeling
- Protein kinase A
- Protein kinase inhibitor
- Recombinant peptides
- Solution NMR