Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban

Cheryl F.C. Ludlam, Isaiah T. Arkin, Xiao Mei Liu, Matthew S. Rothman, Parshuram Rath, Saburo Aimoto, Steven O. Smith, Donald M. Engelman, Kenneth J. Rothschild*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

78 Scopus citations

Abstract

Phospholamban is a 52-amine acid residue membrane protein that regulates Ca2+-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca2+- selective ion channel. We have used polarized attenuated total reflection- Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLS. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm- 1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidylcholine bilayer membranes is predominantly α- helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are α-helical, with an axial orientation that is approximately 30° relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope- labeled hPLB films, our results strongly support models of PLB that predict the existence of an α-helical hydrophobic region spanning the membrane domain.

Original languageAmerican English
Pages (from-to)1728-1736
Number of pages9
JournalBiophysical Journal
Volume70
Issue number4
DOIs
StatePublished - Apr 1996
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by grants from the Army Research Office (ARO) (DAAL03-92-G-0172) and the National Institutes of Health (EY05499 and GM47527) to KJR; from the National Institutes of Health (GM46732) to SOS; and from the National Science Foundation (DMB 8805587) and National Institutes of Health (GM22778) to DME.

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