Cardiolipin containing liposomes are fully ionized at physiological pH. An FT-IR study of phosphate group ionization

Malyshka, Dmitry; Pandiscia, Leah; Schweitzer‐Stenner, Reinhard

doi:10.1016/j.vibspec.2014.10.003

Cited by 34 publications

(42 citation statements)

References 21 publications

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“…It is noteworthy that in this latter study, support for two low pK a values also came from calorimetric experiments. Furthermore, the results of a recent FTIR study with CL-containing liposomes were also consistent with the dual ionization of phosphate groups within a range of pH values (4 to 11) [62]. This study obtained clear spectra for small vesicles composed of 100% CL, but vesicles containing only 20% CL could not be interpreted unambiguously.…”

Section: Discussionsupporting

confidence: 83%

“…A more recent study, based on pH titration and calorimetric measurements with aqueous dispersions of synthetic CL, provided evidence that the CL headgroup behaves as a strong dibasic acid, with both phosphate pK a values between 2 and 3 [61]. Moreover, a recent FTIR analysis of CL-containing liposomes supported the view that the headgroup phosphates were fully ionized at neutral pH [62]. These experimental results are clearly at odds with the dominant model that proposed very different ionization behavior for the two CL phosphates.…”

Section: Introductionmentioning

confidence: 99%

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The ionization properties of cardiolipin and its variants in model bilayers

Sathappa¹,

Alder²

2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The anionic phospholipid cardiolipin has an unusual dimeric structure with a two-phosphate headgroup and four acyl chains. Cardiolipin is present in energy-transducing membranes that maintain electrochemical gradients, including most bacterial plasma membranes and the mitochondrial inner membrane, where it mediates respiratory complex assembly and activation, among many other roles. Dysfunctional biogenesis of cardiolipin is implicated in the pathogenesis of several diseases including Barth syndrome. Because cardiolipin is a dominant anionic lipid in energy-conserving membranes, its headgroup is a major contributor to surface charge density and the bilayer electrostatic profile. However, the proton dissociation behavior of its headgroup remains controversial. In one model, the pKa values of the phosphates differ by several units and the headgroup exists as a monoanion at physiological pH. In another model, both phosphates ionize as strong acids with low pKa values and the headgroup exists in dianionic form at physiological pH. Using independent electrokinetic and spectroscopic approaches, coupled with analysis using Gouy–Chapman–Stern formalism, we have analyzed the ionization properties of cardiolipin within biologically relevant lipid bilayer model systems. We show that both phosphates of the cardiolipin headgroup show strong ionization behavior with low pKa values. Moreover, cardiolipin variants lacking structural features proposed to be required to maintain disparate pKa values – namely the secondary hydroxyl on the central glycerol or a full complement of four acyl chains – were shown to have ionization behavior identical to intact cardiolipin. Hence, these results indicate that within the physiological pH range, the cardiolipin headgroup is fully ionized as a dianion. We discuss the implications of these results with respect to the role of cardiolipin in defining membrane surface potential, activating respiratory complexes, and modulating membrane curvature.

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Section: Discussionsupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

The ionization properties of cardiolipin and its variants in model bilayers

Sathappa¹,

Alder²

2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…On the basis of the above considerations, we propose the following model for the cyt c/CL binding process: in the first step, the positively charged Lys72 and Lys79 facilitate protein-lipid recognition by electrostatic interaction with the deprotonated, negatively charged phosphate groups located on the liposome surface. This view is supported by the recent finding that both the phosphate groups of CL are negatively charged at neutral pH [48]. During this step the protein properly orients on the liposome surface to facilitate the successive step, consisting of a tight protein-lipid binding characterized by the insertion of one (or two [26]) CL acyl chain into the protein interior.…”

Section: Discussionmentioning

confidence: 79%

The key role played by charge in the interaction of cytochrome c with cardiolipin

et al. 2016

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mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys → Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.Abstract Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such Electronic supplementary material The online version of this article

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“…15,16 Despite concurrent work which supported protonation of a CL phosphate in this pH regime, 25 recent studies question whether a CL phosphate ionizes near physiological pH. 26,27 Furthermore, the reverse micelle study, which identified site N, did not detect evidence for a CL binding site near Asn52. 14 Discrepancies in the magnitudes of binding constants for sites A and C also exist in the literature.…”

Section: Introductionmentioning

confidence: 99%

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons

et al. 2016

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Cytochrome c can acquire peroxidase when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7 – 2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6 and ω-undecylenyl-β-D-maltopyranoside bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6 – 4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

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Cardiolipin containing liposomes are fully ionized at physiological pH. An FT-IR study of phosphate group ionization

Cited by 34 publications

References 21 publications

The ionization properties of cardiolipin and its variants in model bilayers

The ionization properties of cardiolipin and its variants in model bilayers

The key role played by charge in the interaction of cytochrome c with cardiolipin

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons

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