Conformation of the cytoplasmic domain of phospholamban by NMR and CD

Abstract

Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to characterize the conformation of the putative cytoplasmic domain of phospholamban (PLB), an oligomeric membrane-bound protein which regulates the activity of the cardiac sarcoplasmic reticulum Ca(2+)-dependent ATPase. In aqueous solution the 25-residue peptide adopts a number of rapidly interconverting conformers with no secondary structural type obviously predominating. However, in trifluoroethanol (TFE) the conformation, while still highly dynamic, is characterized by a high proportion of helical structures. Evidence for this is provided by alpha CH chemical shifts and low NH chemical shift temperature coefficients, small NH-alpha CH intraresidue scalar coupling constants, a substantial number of distinctive interresidue nuclear Overhauser effects (NOEs) [dNN(i, i + 1), d alpha N(i, i + 3), d alpha beta(i, i + 3) and d alpha N(i, i + 4)] and characteristic CD bands at 190 (positive), 206 (negative) and 222 nm (negative). The helicity is interrupted around Pro-21. The activity of PLB is regulated by phosphorylation at either Ser-16 or Thr-17. CD shows that phosphorylation at Ser-16 by the cAMP-activated protein kinase causes about an 11% decrease in alpha-helical content in TFE.

Side Chain and Backbone Dynamics of Phospholamban in Phospholipid Bilayers Utilizing 2H and 15N Solid-State NMR Spectroscopy

“…The principle elements of the chemical shift tensors are represented according to the convention σ 33 ≥ σ 22 ≥ σ 11 . Figure 3 shows the solid-state 2 H NMR powder pattern spectra of site-specific CD 3 -labeled WT-PLB (at residues Leu51, Ala24, and Ala15) and P-PLB (at residue Ala15) incorporated into POPC MLVs.…”

Structure and function of integral membrane protein domains resolved by peptide‐amphiphiles: Application to phospholamban

“…19,37 Isolated cytoplasmic domains of PLB have been observed to be unstructured in aqueous solution, but structured in SDS micelles and TFE. 31,32 Our CD spectra showed that the isolated cytoplasmic domain (PLB 1-25 K) is also unstructured in the presence of lipid bilayers ( Figure 5B). This is, to our knowledge, the first evidence that the isolated cytoplasmic domain of PLB remains unstructured in the presence of lipid bilayers.…”

“…26,30 However, the isolated cytoplasmic domain of PLB is primarily unstructured in aqueous solution, 31 and only forms an ␣-helical conformation when prepared in SDS or trifluoroethanol (TFE) solutions. 31,32 The lack of functional effect observed in the isolated cytoplasmic domain of PLB could be due to a lack of structure in the peptide, the low affinity of the free cytoplasmic peptide for the Ca-ATPase, or incorrect positioning of the peptide. In order for the cytoplasmic domain of PLB to have a functional effect on the Ca-ATPase, it is likely than the peptide must be anchored to the membrane surface in a configuration that optimizes the potential for interaction with the Ca-ATPase.…”

De novo design of a pentameric coiled‐coil: decoding the motif for tetramer versus pentamer formation in water‐soluble phospholamban*

“…2A). This finding was not surprising, given that in both PLB and WSPLB residues 1–25 are essentially unstructured in the absence of detergents or lipids (30–34). A recent NMR structure of the monomeric mutant C41F of PLB in DPC micelles by Zamoon et al (35) revealed that residues 2–13 are in fact α ‐helical in the presence of dodecylphospholcholine (DPC) detergents.…”