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Molecular Biology

2011

NMR

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Camp-Dependent Protein Kinase A Selects The Excited State Of The Membrane Substrate Phospholamban, Larry R. Masterson Aug 2011

Camp-Dependent Protein Kinase A Selects The Excited State Of The Membrane Substrate Phospholamban, Larry R. Masterson

Larry Masterson

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca2+-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, …

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Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch Jan 2011

Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch

Lila Gierasch

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including …

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