Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone

Christine B. Karim, Tara L. Kirby, Zhiwen Zhang, Yuri Nesmelov, David D. Thomas

Research output: Contribution to journalArticlepeer-review

103 Scopus citations

Abstract

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the α-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4- carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg2+ shifts it in the opposite direction. Relaxation enhancement, due to Ni2+ ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.

Original languageEnglish (US)
Pages (from-to)14437-14442
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number40
DOIs
StatePublished - Oct 5 2004

Fingerprint Dive into the research topics of 'Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone'. Together they form a unique fingerprint.

Cite this