Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.
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 (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.
Masterson
Larry R
LR
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Bortone
Nadia
N
Yu
Tao
T
Ha
Kim N
KN
Gaffarogullari
Ece C
EC
Nguyen
Oanh
O
Veglia
Gianluigi
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C518603:protein kinase inhibitor peptide (5-24); D017868:Cyclic AMP-Dependent Protein Kinases