Abstract
Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Esherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 253-261 |
| Number of pages | 9 |
| Journal | Protein Expression and Purification |
| Volume | 30 |
| Issue number | 2 |
| DOIs | |
| State | Published - Aug 1 2003 |
Bibliographical note
Funding Information:The authors thank Z. Zhang for assistance in synthesizing and purifying the synthetic proteins, Becky Eggimann for running the NMR experiments on sarcolipin, and D. Live and B. Ostrowski for assisting with the NMR experiments, T. Krick and Dr. L.A. Higgins for assistance in the Facility for Mass Spectrometry in the Life Sciences at the University of Minnesota, J. Johnson and Dr. L. Dangott for assistance in amino acid analysis of synthetic AFA-PLB at the Protein Chemistry Laboratory, Texas A&M University, and R. Haase and F. Konstantinides for assistance in amino acid analysis of recombinant AFA-PLB at the Scientific Research Consortium. We benefited from the outstanding facilities at the Minnesota High-Field NMR Center in the Department of Biochemistry, Molecular Biology, and Biophysics; and the Solid State NMR facility in the Department of Chemistry, University of Minnesota. NMR instrumentation was provided with funds from the NSF (BIR-961477) and the University of Minnesota Medical School.
Keywords
- Maltose binding protein
- Membrane protein overexpression
- Micelles
- Recombinant phospholamban
- Recombinant sarcolipin
- Solid-state NMR
- Solution NMR