Received November 24, 1999

Revised Manuscript Received August 25, 2000

Abstract:

Phospholamban is an integral membrane protein having a single membrane-spanning helix which forms a pentameric complex in cardiac and smooth muscle cell membranes. Deuterium NMR measurements of leucine residues in the transmembrane domain of the protein provide a novel approach for establishing the rotational orientation of the phospholamban monomer within the complex. At 5 C, the spectra of Leu43 and Leu44 are similar and exhibit a quadrupole splitting of 33 kHz. This splitting is slightly narrower than the ~40 kHz splitting which results solely from rapid methyl group rotation. The deuterium line shape of Leu42 has lost the distinctive 33-kHz quadrupole splitting due to increased librational motion of the side chain and/or rotation about the C-C and C-C bonds. The observed line shapes of the three consecutive leucine residues in phospholamban are consistent with Leu42 being oriented toward the lipids, where it exhibits fewer steric contacts, and Leu43 and Leu44 being oriented toward helix interfaces which restrict their motion. Possible packing arrangements of the three transmembrane leucine residues in the phospholamban pentamer are examined using computational methods to assess the packing restrictions of the leucine side chains. The results are discussed in terms of models of the phospholamban pentamer previously proposed on the basis of mutational data.

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