Spectroscopic studies of the interaction of Ca²⁺‐ATPase‐peptides with dodecyl maltoside and its brominated analog

Abstract

The transmembrane sector of sarcoplasmic reticulum Ca 2ϩ -ATPase comprises ten putative transmembrane spans (M1ϪM10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca 2ϩ binding: peptide M6 (amino acid residues 785Ϫ810), peptide M7-L (amino acid residues 808Ϫ847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818Ϫ847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the A-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca 2ϩ -ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl β- D-maltoside Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl β-D-maltoside/ 7,8-dibromododecyl β-maltoside, using tryptophan octyl ester and solubilized Ca 2ϩ -ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca 2ϩ -ATPase 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 Ca 2ϩ -ATPase and the rather short A-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca 2ϩ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca 2ϩ binding.

Tryptophan Octyl Ester in Detergent Micelles of Dodecylmaltoside: Fluorescence Properties and Quenching by Brominated Detergent Analogs

“…The binding of TOE to DM micelles led to a large (maximum eight times) increase in TOE fluorescence intensity (Fig. 2 A; see also Soulié et al, 1998), which was correlated with a blue shift in the emission spectrum. These changes may result from a significant decrease in the polarity (and/or mobility) of the TOE microenvironment upon binding to detergent micelles.…”

“…These brominated detergents were also used to detect conformational changes in solubilized mutants (with a single Trp) of another membrane protein, the lactose permease from Escherichia coli (Wang et al, 1997). Use of these detergents was also extended to the study of the interaction between detergent and peptides corresponding to transmembrane segments of the SR Ca 2ϩ -ATPase (Soulié et al, 1998).…”

Involvement of the Cytoplasmic Loop L6–7 in the Entry Mechanism for Transport of Ca2+ through the Sarcoplasmic Reticulum Ca2+-ATPase

“…These were the same peptides as previously used in a peptide study on micelle insertion as a model of membrane interaction (37). Ca 2ϩ binding of these and the L6 -7 peptides was measured with the aid of an Aminco double-beam spectrophotometer, using murexide as an indicator of the concentration of free Ca 2ϩ .…”

“…The formation of the Ca 2ϩ -murexide complex was recorded from the increase in absorption of 0.2 mM murexide at 544 nm, relative to that at 495 nm. To measure Ca 2ϩ binding by M7-L and M7-S, the peptides (1 mg/ml) were first solubilized in SDS (10 mg/ml; these peptides are known to be monomeric in SDS (37)). Furthermore, peptide binding data were corrected from the decrease in free concentration of Ca 2ϩ resulting from the interaction with the SDS micelles as measured in the absence of peptide.…”

Detergents as Probes of Hydrophobic Binding Cavities in Serum Albumin and Other Water-Soluble Proteins

“…8. This compound has previously proved useful to study tryptophan accessibility in membranous and solubilized sarcoplasmic reticulum Ca 2ϩ -ATPase (de Foresta et al, 1996) and in Ca 2ϩ -ATPase peptide-detergent complexes (Soulié et al, 1998). Quenching occurs primarily by collisional contacts of bromine atoms with tryptophan, but also has a partly static character under conditions of high medium viscosity (de Foresta et al, 1999).…”