SPECTROSCOPIC STUDIES OF THE INTERACTION OF CA2-ATPASE-PEPTIDES WITH DODECYL MALTOSIDE AND ITS BROMINATED ANALOG()

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase compris es ten putative transmembrane spans (M1-M10) in current topology model s. We report here the structure and properties of three synthetic pept ides with a single Trp representing the M6 and M7 regions implicated i n Ca2+ binding: peptide M6 (amino acid residues 785 - 810), peptide M7 -L (amino acid residues 808 - 847) corresponding to loop 6-7 and the m ajority of span M7, and peptide M7-S (amino acid residues 818-847) whi ch contains a shorter version of loop 6-7 than M7-L. After uptake of t he peptides ill the hydrophobic environment of dodecyl maltoside micel les, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would de expected for a classical transmembrane segme nt. For M7-L peptide, the L6-7 loop is subject to specific: proteolyti c cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluor escence intensity changes, either enhancement using n-dodecyl beta-D-m altoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de F oresta, B., Legros, N., Plusquellec. D., le Maire, M. & Champeil, P. ( 1996) Eur. J. Biochem. 241, 343-354]. Our results indicate that M7-L a nd M7-S are completely taken rtp by the detergent micelles. In contras t, the M6 peptide, which is highly water soluble, is more loosely asso ciated with the detergent, as is also demonstrated by size-exclusion c hromatography. The location of Trp ill micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7, 8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solu bilized Ca2+-ATPase as reference compounds. We conclude that W832 in M 7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPa se topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short a-helix span of M6 may lead to exposure of a significant part of the C-terminal of th is peptide to the micelle surface. The results are discussed in relati on to the proposed roles of these membrane segments in active transpor t of Ca2+ ions, in particular, the demonstration that M6 does not beha ve as a classical transmembrane helix may be correlated with the evide nce, from site-directed mutagenesis, that this transmembrane segment s hould be essential in Ca2+ binding.