Two-Dimensional Solid-State NMR Reveals Two Topologies of Sarcolipin in Oriented Lipid Bilayers

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• 作者：Jarrod J. Buffy ; Nathaniel J. Traaseth ; Alessandro Mascioni ; Peter L. Gor'kov ; Eduard Y. Chekmenev ; William W. Brey ; Gianluigi Veglia
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：September 12, 2006
• 年：2006
• 卷：45
• 期：36
• 页码：10939 - 10946
• 全文大小：184K
• 年卷期：v.45,no.36(September 12, 2006)
• ISSN：1520-4995

文摘

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a andSERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca²⁺ affinityand thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch musclefibers with significant expression levels also found in the cardiac muscle. SLN shares ~30% identitywith the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried outin detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1Dsolid-state NMR experiments on selectively ¹⁵N-labeled sites showed that SLN crosses the lipid bilayerwith an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLNstructure and its interactions with both lipids and SERCA, herein we report our initial structural andtopological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D¹⁵N PISEMA experiments. The PISEMA spectra obtained on uniformly ¹⁵N-labeled protein as well as¹⁵N-Leu, ¹⁵N-Ile and ¹⁵N-Val map the secondary structure of SLN and, simultaneously, reveal that SLNexists in two distinct topologies. Both the major and the minor populations assume an orientation withthe helix axis tilted by ~23 with respect to the lipid bilayer normal, but vary in the rotation angle aboutthe helix axis by ~5. The existence of the multiple populations in model membranes may be a significantrequirement for SLN interaction with SERCA.