Expression and purification of an isotopically labeled aggregation prone inducible nitric oxide synthase calmodulin-binding protein for use in nuclear magnetic resonance studies
✍ Scribed by Michael Piazza; Yay Duangkham; Donald E. Spratt; Thorsten Dieckmann; J. Guy Guillemette
- Publisher
- John Wiley and Sons
- Year
- 2011
- Tongue
- French
- Weight
- 250 KB
- Volume
- 54
- Category
- Article
- ISSN
- 0022-2135
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✦ Synopsis
Nuclear magnetic resonance (NMR) spectroscopy is an efficient method for studying the dynamics and structures of protein‐protein and protein‐peptide complexes. Calmodulin (CaM), a ubiquitous Ca^2+^‐sensing protein, is able to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. The investigation of CaM bound to the CaM‐binding region of the inducible nitric oxide synthase (iNOS) isoform of NOS proved to be difficult due to the propensity of the iNOS CaM‐binding domain to aggregate when not bound to CaM. In the present study, an isotopically labeled peptide of the CaM‐binding region of iNOS has successfully been expressed and purified. The peptide identity was verified by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy and further characterized by NMR spectroscopy. These results demonstrate an efficient approach for the expression and purification of individually stable isotope labeled protein complexes for NMR analysis, even if one partner is prone to aggregation or has very low solubility. This technique can also be used to label a specific type of amino acid or radiolabel all of the amino acids in one of the components participating in a protein–protein or protein–peptide complex.