Philippe Champeil

Interaction of membrane proteins and lipids with solubilizing detergents

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins.

Calcium control on InsP3-induced discharge of calcium from permeabilised hepatocyte pools

The control exerted by intralumenal and cytosolic Ca2+ on InsP3-induced release of Ca2+ from intracellular Ca2+ pools in suspensions of saponin-permeabilised rat hepatocytes was investigated by combined Quin-2 and 45Ca2+ measurements at 20 degrees C. We failed to detect a major effect of intralumenal Ca2+ in regulating this release, as various manipulations in which the load of the Ca2+ pools was varied by a factor of two did not significantly affect the apparent relative efficiency of InsP3 in releasing Ca2+; these manipulations included loading the Ca2+ pools up to various steady state levels by preliminary equilibration at various external free Ca2+ concentrations, as well as emptying them progressively through the blockade of pump-mediated Ca2+ uptake. As regards Ca2+ on the cytosolic side, in contrast with recent results obtained with other systems, we found that, at maximal doses, InsP3-induced Ca2+ release was not stimulated by raising Ca2+ from very low to submicromolar or micromolar concentrations, and that only relatively high concentrations of free Ca2+ inhibited this release (half-maximal inhibition was between 3 and 15 microM). Such elevated Ca2+ concentrations reduced the size of the InsP3-sensitive Ca2+ pool. We also noted that the apparent cooperativity of InsP3 activation of release at pCa 5 was noticeably less than that observed at pCa 7. As a result, at low InsP3 concentrations, a rise in cytosolic Ca2+ from pCa 7 to pCa 5 stimulated InsP3-mediated Ca2+ release. These results are discussed in the context of the current speculations about tissue specificity, heterogeneity, quantal release, oscillations, and the several different mechanisms that may control InsP3-induced Ca2+ release.

Does a radiolabelled ligand bind to a homogeneous population of non-interacting receptor sites?

Receptor subtypes of pharmacological interest are often characterized by studies in which a particular radiolabelled ligand is competitively displaced from its binding sites, either by its unlabelled analogue (homologous displacement), or by different, unlabelled, ligands (heterologous displacement). In this article, Stéphane Swillens, Magali Waelbroeck and Philippe Champeil emphasize the fact that a single homologous displacement curve is generally unable to reveal that the radioligand binds to a heterogeneous receptor population. Overlooking this heterogeneity may, in turn, dramatically affect interpretation of heterologous displacement curves displaying more than one receptor type for the unlabelled ligand: neither the binding affinities of the ligands for the different receptors, nor the proportion of these receptors, can be estimated reliably.

Crystal Structure of D351A and P312A Mutant Forms of the Mammalian Sarcoplasmic Reticulum Ca2+-ATPase Reveals Key Events in Phosphorylation and Ca2+ Release

In recent years crystal structures of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{AlF}_{4}^{-}\) \end{document} bound (3.5-Å resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-Å resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca2+ was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca2+-translocating Ca2E1P→E2P transition, which seems to be due to a stabilization of Ca2E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro312 with alanine in P312A.

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

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.

Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum?

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium‐free medium containing magnesium and calcium, at pH 6 and 20°C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane‐bound ionophore A23187, the observed turnoverdependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.

Effects of Inhibitors on Luminal Opening of Ca2+ Binding Sites in an E2P-like Complex of Sarcoplasmic Reticulum Ca22+-ATPase with Be22+-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative “E2P-related” complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the “ADP-insensitive” phosphorylated form of Ca2+-ATPase, the so-called “E2P” state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.

Phosphatidylserine Stimulation of Drs2p·Cdc50p Lipid Translocase Dephosphorylation Is Controlled by Phosphatidylinositol-4-phosphate

Background: Transport of phosphatidylserine (PS) analogs by the Drs2p flippase is regulated by PtdIns(4)P. Results: PS stimulates dephosphorylation of the Drs2p·Cdc50p complex only in the presence of PtdIns(4)P. Conclusion: The step at which PtdIns(4)P regulates lipid transport is identified. Significance: Our coordinated overexpression system provides mechanistic insight into PS transport and will be useful for further Drs2p characterization and crystallization. Here, Drs2p, a yeast lipid translocase that belongs to the family of P4-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-32P]ATP of a 32P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here.

Characterization of the co-agonist effects of strontium and calcium on myo-inositol trisphosphate-dependent ion fluxes in cerebellar microsomes.

Using sheep cerebellum microsomes previously loaded with 45Ca2+ or 90Sr2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP3)-induced efflux of these ions on Ca2+ or Sr2+ on the cytosolic side. At a low InsP3 concentration, Ca2+ in the submicromolar range only poorly activated 45Ca2+ or 90Sr2+ efflux, and higher Ca2+ concentrations were inhibitory. In contrast, Sr2+ in the micromolar range activated release efficiently, while only very high Sr2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP3 concentration, which allowed increasing free Ca2+ to micromolar concentrations without inducing complete inhibition of the InsP3-dependent efflux. Under these conditions, micromolar Ca2+ was found to activate efflux to a large extent, similar to that previously found with Sr2+. Optimal activation by Ca2+ of the InsP3-dependent channel occurs at micromolar rather than submicromolar free Ca2+ concentrations, but at too low an InsP3 concentration, Ca(2+)-induced activation is counteracted by Ca(2+)-induced inactivation. Separate measurements of [3H]-InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]-InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptors affinity for InsP3. A similar relationship also holds in the case of the activating effect of nucleotides.

31P NMR as a tool for monitoring detergent solubilization of sarcoplasmic reticulum membranes

Sarcoplasmic reticulum vesicles were solubilized stepwise by the nonionic detergent octaethyleneglycol monododecyl ether; 31P NMR enabled the extent of phospholipid solubilization to be monitored by following the conversion of the broad resonance peak characterizing the phospholipids inserted in the bilayer to the narrow resonance signal characterizing phospolipids inserted into a mixed micelle. Up to 0.25 g detergent/g protein could be incorporated into the membrane without solubilization. Higher detergent concentrations of up to 1.5–2 g detergent/g protein led to gradual solubilization. Although the method allows us to monitor the extent of solubilization of individual phospholipid classes, there was no evidence of either preferential solubilization or retention of a specific class of phospholipids.