Recent Research Developments

Emily Metcalfe from the Veglia laboratory has elucidated the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using ¹H/¹⁵N heteronuclear NMR spectroscopy. Phospholamban is a 52 amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 – 52, a connecting loop from 17 – 21, and a cytoplasmic domain from 1 – 16 that is organized in an �gL-shaped�h structure where the transmembrane and the cytoplasmic domain form an angle of approximately 80 degrees (Mascioni et al JACS 2002; Zamoon et al Biophys J. 2003). T₁, T₂ and ¹H/¹⁵N NOE values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22 - 30). In addition, using CPMG (Carr-Purcell-Meiboom-Gill)-based experiments, Emily characterized phospholamban dynamics in the ms-ms time scale. Emily found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the mms-ms range, while she detected minimal dynamics for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and in the cytoplasmic helix. These dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.

Emily�fs paper will be published in the August issue of Biophys J.

Figure: Modelfree analysis of ¹⁵N relaxation data of AFA-PLB. Order parameter (S²) (top), internal correlation time (t_e) (middle) and exchange terms (Rex) (Bottom) plotted as a function of the residue number for AFA-PLB. The four dynamics domains are color coded and mapped onto a representative structure of PLB determined in micelles using solution NMR experiments. The color scheme is blue, S²�†0.85, red, S²�†0.75, yellow, S²<0.75 (Zamoon et al 2003).

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