Kinetics and thermodynamics of association of a phospholipid derivative with lipid bilayers in liquid-disordered and liquid-ordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) in liquid-disordered and liquid-ordered phase lipid bilayer membranes, of the fluorescent phospholipid derivative NBD-dimyristoylphosphatidyl ethanolamine. This study made use of a recently described method that exploits a detailed knowledge of the binding kinetics of an amphiphile to bovine serum albumin, to recover the insertion and desorption rate constants when the albumin-bound amphiphile is transferred through the aqueous phase to the membrane and vice versa. The lipid bilayers, studied as large unilamellar vesicles, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine in the liquid-disordered phase; and from two cholesterol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and cholesterol (molar ratio of 1:1), and egg sphingomyelin and cholesterol (molar ratio of 6:4), both in the liquid-ordered phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were directly measured between 15 degrees and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes, were derived from this data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and permitted the estimation of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane.     

Kinetics and thermodynamics of association of a fluorescent lysophospholipid derivative with lipid bilayers in liquid-ordered and liquid-disordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) of the fluorescent lysophospholipid derivative NBD-lyso-1-myristoylphosphatidylethanolamine in l(d) and l(o) phase lipid bilayer membranes. The lipid bilayers, studied as LUV, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine, in the l(d) phase; and from two Chol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and Chol (molar ratio of 1:1) and SpM and Chol (molar ratio of 6:4), both in the l(o) phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were measured between 15 degrees C and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes were derived from these data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and an estimation was made of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane. We show that, contrary to general belief, the association of NBD-lysoMPE with lipid bilayers is not a diffusion-controlled process, the rate-limiting step in insertion being the formation of a free area in the membrane surface of an adequate size for insertion to occur.     

Partitioning of lipidated peptide sequences into liquid-ordered lipid domains in model and biological membranes.

We have used a fluorescence assay and detergent fractionation to examine the partitioning of different fluorescent lipidated peptides, with sequences and lipid substituents matching those found in various classes of lipidated cellular proteins, into liquid-ordered (raft-like) domains in lipid bilayers. Peptides incorporating isoprenyl groups, or multiple unsaturated acyl chains, show negligible affinity for liquid-ordered domains in mixed-phase liquid-ordered/liquid-disordered (l(o)/l(d)) bilayers composed of dipalmitoylphosphatidylcholine, a spin-labeled unsaturated phosphatidylcholine, and cholesterol. By contrast, peptides incorporating multiple S- and/or N-acyl chains, or a cholesterol residue plus an N-terminal palmitoyl chain, show significant partitioning into liquid-ordered domains under the same conditions. Interestingly, the affinity of a lipidated peptide for l(o) domains can be strongly influenced, not only by the structures of the lipid substituents but also by the nature and the positions of their attachment to the peptide chain. These results are well correlated with those obtained from parallel assays based on low-temperature detergent fractionation. Using the latter approach, we further demonstrate that a truly minimal l(o) domain partitioning motif [myristoylGlyCys(palmitoyl)-] can mediate efficient incorporation into the "raft" fraction of COS-7 cell membranes.     

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.     

Cholesterol does not induce segregation of liquid-ordered domains in bilayers modeling the inner leaflet of the plasma membrane

A fluorescence-quenching method has been used to assess the potential formation of segregated liquid-ordered domains in lipid bilayers combining cholesterol with mixtures of amino and choline phospholipids like those found in the cytoplasmic leaflet of the mammalian cell plasma membrane. When present in proportions >20-30 mol %, different saturated phospholipids show a strong proclivity to form segregated domains when combined with unsaturated phospholipids and cholesterol, in a manner that is only weakly affected by the nature of the phospholipid headgroups. By contrast, mixtures containing purely unsaturated phospholipids and cholesterol do not exhibit detectable segregation of domains, even in systems whose components differ in headgroup structure, mono- versus polyunsaturation and/or acyl chain heterogeneity. These results indicate that mixtures of phospholipids resembling those found in the inner leaflet of the plasma membrane do not spontaneously form segregated liquid-ordered domains. Instead, our findings suggest that factors extrinsic to the inner-monolayer lipids themselves (e.g., transbilayer penetration of long sphingolipid acyl chains) would be essential to confer a distinctive, more highly ordered organization to the cytoplasmic leaflet of "lipid raft" structures in animal cell membranes.     

Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes

We present a detailed study of the translocation rate of two headgroup-labeled phospholipid derivatives, one with two acyl chains, NBD-DMPE, and the other with a single acyl chain, NBD-lysoMPE, in lipid bilayer membranes in the liquid-disordered state (POPC) and in the liquid-ordered states (POPC/cholesterol (Chol), molar ratio 1:1, and sphingomyelin (SpM)/Chol, molar ratio 6:4). The study was performed as a function of temperature and the thermodynamic parameters of the translocation process have been obtained. The most important findings are 1), the translocation of NBD-DMPE is significantly faster than the translocation of NBD-lysoMPE for all bilayer compositions and temperatures tested; and 2), for both phospholipid derivatives, the translocation in POPC bilayers is approximately 1 order of magnitude faster than in POPC/Chol (1:1) bilayers and approximately 2-3 orders of magnitude faster than in SpM/Chol (6:4) bilayers. The permeability of the lipid bilayers to dithionite has also been measured. In liquid disordered membranes, the permeability rate constant obtained is comparable to the translocation rate constant of NBD-DMPE. However, in liquid-ordered bilayers, the permeability of dithionite is significantly faster then the translocation of NBD-DMPE. The change in enthalpy and entropy associated with the formation of the activated state in the translocation and permeation processes has also been obtained.     

Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains

Synaptotagmin I is a synaptic vesicle associated membrane protein that appears to regulate Ca(2+)-mediated exocytosis. Here, the Ca(2+)-dependent membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the membrane orientation and position of synaptotagmin at the bilayer interface. Both C2A and C2B bind to the membrane interface with their first and third Ca(2+) binding loops penetrating the membrane interface. The polybasic face of C2B does not interact with the membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the isolated domains, both C2A and C2B have similar orientations; however, the two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently but influence their mutual membrane penetration. This may explain the occurrence of multiple C2 domains in proteins that function in membrane trafficking and repair.     

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

Ca²⁺ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca²⁺ in triggering vesicle fusion though the Ca²⁺ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca²⁺ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca²⁺–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca²⁺-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca²⁺ sensor for presynaptic vesicle fusion.     

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.     

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.     

Detection of apoptosis using the C2A domain of synaptotagmin I

Binding of annexin V or the C2A domain of synaptotagmin I to phosphatidylserine expressed on the surface of apoptotic cells can, when labeled with appropriate probe molecules, be used to detect the presence of apoptosis using radionuclide, magnetic resonance, and optical imaging techniques. The preparation of a biotinylated C2A-GST fusion protein is described, and its capability, when used in conjunction with fluorescein-labeled streptavidin, of detecting apoptotic cells by flow cytometry is compared directly with the performance of a commercial preparation of fluorescein-labeled annexin V. Biotinylated C2A-GST, when used in conjunction with streptavidin-conjugated superparamagnetic iron oxide nanoparticles or Gd-chelate-avidin conjugates, was shown to be capable of detecting apoptotic cells using T(2)-weighted or T(1)-weighted magnetic resonance imaging experiments, respectively.     

Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I

Synaptotagmin I (Syt I), an evolutionarily conserved integral membrane protein of synaptic vesicles, is now known to regulate Ca2+-dependent neurotransmitter release. Syt I protein should undergo several post-translational modifications before maturation and subsequent functioning on synaptic vesicles (e.g. N-glycosylation and fatty acylation in vertebrate Syt I), because the apparent molecular weight of Syt I on synaptic vesicles (mature form, 65,000) was much higher than the calculated molecular weight (47,400) predicted from the cDNA sequences both in vertebrates and invertebrates. Common post-translational modification(s) of Syt I conserved across phylogeny, however, have never been elucidated. In the present study, I discovered that dithreonine residues (Thr-15 and Thr-16) at the intravesicular domain of mouse Syt I are post-translationally modified by a complex form of O-linked sugar (i.e. the addition of sialic acids) in PC12 cells and that the O-glycosylation of Syt I in COS-7 cells depends on the coexpression of vesicle-associated membrane protein-2 (VAMP-2)/synaptobrevin. I also showed that a transmembrane domain of Syt I directly interacts with isolated VAMP-2, but not VAMP-2, in the heterotrimeric SNARE (SNAP receptor) complex (vesicle SNARE, VAMP-2, and two target SNAREs, syntaxin IA and SNAP-25). Since di-Thr or di-Ser residues are often found at the intravesicular domain of invertebrate Syt I, and VAMP-dependent O-glycosylation was also observed in squid Syt expressed in COS-7 cells, I propose that VAMP-dependent O-glycosylation of Syt I is a common modification during evolution and may have important role(s) in synaptic vesicle trafficking.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I

Synaptotagmin I (syt), a transmembrane protein localized to secretory vesicles, functions as a Ca2+ sensor that facilitates SNARE-mediated membrane fusion. The cytoplasmic domain of syt harbors two C2-domains designated C2A and C2B. Upon binding Ca2+, C2A and C2B partially penetrate into membranes that contain anionic phospholipids. However, it is unknown whether these tandem C2-domains engage membranes at the same time, in a sequential manner, or in a mutually exclusive manner. We have used site-directed fluorescent probes to monitor the penetration of syt's C2-domains into phosphatidylserine-harboring lipid bilayers. We report that, in response to Ca2+, C2A and C2B copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more efficient when synthetic rather than natural phospholipids were used to prepare bilayers. The membrane penetration activity of the intact cytoplasmic domain of syt (C2A-C2B) exhibits significant resistance to changes in ionic strength. In contrast, the ability of isolated C2B to bind membranes in response to Ca2+ can be disrupted by subtle changes in ionic strength. Tethering C2B to a mutant version of C2A that does not bind Ca2+ or membranes significantly increases the stability of Ca2+.C2B.membrane complexes, confirming that C2A affects the membrane-binding properties of the adjacent C2B domain.     

Membrane-bound orientation and position of the synaptotagmin I C2A domain by site-directed spin labeling

Site-directed spin labeling was used to determine the membrane orientation and insertion of the C2A domain from synaptotagmin I. A series of single cysteine mutants of the C2A domain of synaptotagmin I was prepared and labeled with a sulfhydryl specific spin label. Upon Ca2+ or membrane binding, the EPR line shapes of these mutants reveal dramatic decreases in label mobility within the Ca2+-binding loops. This loss in mobility is likely due in part to a reduction in local backbone fluctuations within the loop regions. Power saturation was then used to determine the position of each spin-labeled site along the bilayer normal, and these EPR distance constraints were used along with the high-resolution solution structure of C2A to generate a model for the orientation and position of the domain at the membrane interface. This model places the polypeptide backbone of both the first and third Ca2+-binding loops in contact with the membrane interface, with several labeled side chains lying within the bilayer interior. All three Ca2+-binding sites lie near a plane defined by the lipid phosphates. This model indicates that there is some desolvation of this domain upon binding and that hydrophobic as well as electrostatic interactions contribute to the binding of C2A. When compared to the C2 domain from cPLA2 (Frazier et al. (2002) Biochemistry 41, 6282), a similar orientation for the beta-sandwich region is found; however, the cPLA2 C2 domain is translocated 5-7 A deeper into the membrane hydrocarbon. This difference in depth is consistent with previous biophysical data and with the difference that long-range electrostatic interactions and desolvation are expected to make to the binding of these two C2 domains.