Structure and fluctuations of charged phosphatidylserine bilayers in the absence of salt

Using x-ray diffraction and NMR spectroscopy, we present structural and material properties of phosphatidylserine (PS) bilayers that may account for the well documented implications of PS headgroups in cell activity. At 30 degrees C, the 18-carbon monounsaturated DOPS in the fluid state has a cross-sectional area of 65.3 A(2) which is remarkably smaller than the area 72.5 A(2) of the DOPC analog, despite the extra electrostatic repulsion expected for charged PS headgroups. Similarly, at 20 degrees C, the 14-carbon disaturated DMPS in the gel phase has an area of 40.8 A(2) vs. 48.1 A(2) for DMPC. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. Unlike zwitterionic lipids, stacks of PS bilayers swell indefinitely as water is added. Data obtained for osmotic pressure versus interbilayer water spacing for fluid phase DOPS are well fit by electrostatic interactions calculated for the Gouy-Chapman regime. It is shown that the electrostatic interactions completely dominate the fluctuational pressure. Nevertheless, the x-ray data definitively exhibit the effects of fluctuations in fluid phase DOPS. From our measurements of fluctuations, we obtain the product of the bilayer bending modulus K(C) and the smectic compression modulus B. At the same interbilayer separation, the interbilayer fluctuations are smaller in DOPS than for DOPC, showing that B and/or K(C) are larger. Complementing the x-ray data, (31)P-chemical shift anisotropy measured by NMR suggest that the DOPS headgroups are less sensitive to osmotic pressure than DOPC headgroups, which is consistent with a larger K(C) in DOPS. Quadrupolar splittings for D(2)O decay less rapidly with increasing water content for DOPS than for DOPC, indicating greater perturbation of interlamellar water and suggesting a greater interlamellar hydration force in DOPS. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The ¹³C R₁ and ¹³C-¹H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the α-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Thermodynamics of the coil <==> beta-sheet transition in a membrane environment

Biologically important peptides such as the Alzheimer peptide Abeta(1-40) display a reversible random coil <==>beta-structure transition at anionic membrane surfaces. In contrast to the well-studied random coil left arrow over right arrow alpha-helix transition of amphipathic peptides, there is a dearth on information on the thermodynamic and kinetic parameters of the random coil left arrow over right arrow beta-structure transition. Here, we present a new method to quantitatively analyze the thermodynamic parameters of the membrane-induced beta-structure formation. We have used the model peptide (KIGAKI)(3) and eight analogues in which two adjacent amino acids were substituted by their d-enantiomers. The positions of the d,d pairs were shifted systematically along the three identical segments of the peptide chain. The beta-structure content of the peptides was measured in solution and when bound to anionic lipid membranes with circular dichroism spectroscopy. The thermodynamic binding parameters were determined with isothermal titration calorimetry and the binding isotherms were analysed by combining a surface partition equilibrium with the Gouy-Chapman theory. The thermodynamic parameters were found to be linearly correlated with the extent of beta-structure formation. beta-Structure formation at the membrane surface is characterized by an enthalpy change of DeltaH(beta)=-0.23 kcal/mol per residue, an entropy change of DeltaS(beta)=-0.24 cal/mol K residue and a free energy change of DeltaG(beta)=-0.15 kcal/mol residue. An increase in temperature induces an unfolding of beta-structure. The residual free energy of membrane-induced beta-structure formation is close to that of membrane-induced alpha-helix formation.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: A spectroscopic and polarity mismatch study

The V-ATPases are a family of ATP-dependent proton pumps, involved in a variety of cellular processes, including bone breakdown. V-ATPase enzymes that are too active in the latter process can result in osteoporosis, and inhibitors of the enzyme could be used to treat this disease. As a first step in studying the structure and function of the membrane-embedded interface at which proton translocation takes place, and its role in V-ATPase inhibition, synthetic peptides P1 and P2 consisting of 25 amino acid residues are presented here that mimic Vph1p helix 7 of yeast V-ATPase. A single mutation R10A between peptide P1 and P2 makes it possible to focus on the role of the essential arginine residue R735 in proton translocation. In the present work, we use a novel combination of spectroscopic techniques, such as CD spectroscopy, tryptophan emission spectra, acrylamide quenching and parallax analysis, and polarity mismatch modeling to characterize the peptides P1 and P2 in lipid bilayer systems. Based on both the spectroscopic experiments and the polarity mismatch modeling, P1 and P2 adopt a similar transmembrane conformation, with a mainly α-helical structure in the central part, placing the tryptophan residue at position 12 at a location 4 ± 2 Å from the centre of the lipid bilayer. Furthermore, the arginine at position 10 in P1 does not have an effect on the bilayer topology of the peptide, showing that the long, flexible side chain of this residue is able to snorkel towards the lipid headgroup region. This large flexibility of R735 might be important for its function in proton translocation in the V-ATPase enzyme.     

Component volumes of unsaturated phosphatidylcholines in fluid bilayers: a densitometric study

The specific volumes of six 1,2-diacylphosphatidylcholines with monounsaturated acyl chains (diCn:1PC, n=14-24 is the even number of acyl chain carbons) in fluid bilayers in multilamellar vesicles dispersed in H(2)O were determined by the vibrating tube densitometry as a function of temperature. From the data obtained with diCn:1PC (n=14-22) vesicles in combination with the densitometric data from Tristram-Nagle et al. [Tristram-Nagle, S., Petrache, H.I., Nagle, J.F., 1998. Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers. Biophys. J. 75, 917-925.] and Koenig and Gawrisch [Koenig, B.W., Gawrisch, K., 2005. Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase. Biochim. Biophys. Acta 1715, 65-70.], the component volumes of phosphatidylcholines in fully hydrated fluid bilayers at 30 degrees C were obtained. The volume of the acyl chain CH and CH(2) group is V(CH)=22.30 A(3) and V(CH2) =A(3), respectively. The volume of the headgroup including the glyceryl and acyl carbonyls, V(H), and the ratio of acyl chain methyl and methylene group volumes, r=V(CH3):V(CH2) are linearly interdependent: V(H)=a-br, where a=434.41 A(3) and b=-55.36 A(3) at 30 degrees C. From the temperature dependencies of component volumes, their isobaric thermal expansivities (alpha(X)=V(X)(-1)(partial differential V(X)/ partial differential T) where X=CH(2), CH, or H were calculated: alpha(CH2)=118.4x10(-5)K(-1), alpha(CH)=71.0x10(-5)K(-1), alpha(H)=7.9x10(-5)K(-1) (for r=2) and alpha(H)=9.6x10(-5)K(-1) (for r=1.9). The specific volume of diC24:1PC changes at the main gel-fluid phase transition temperature, t(m)=26.7 degrees C, by 0.0621 ml/g, its specific volume is 0.9561 and 1.02634 ml/g at 20 and 30 degrees C, respectively, and its isobaric thermal expansivity alpha=68.7x10(-5) and 109.2x10(-5)K(-1) below and above t(m), respectively. The component volumes and thermal expansivities obtained can be used for the interpretation of X-ray and neutron scattering and diffraction experiments and for the guiding and testing molecular dynamics simulations of phosphatidylcholine bilayers in the fluid state.     

Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel α-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 °C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH₂, CH, and terminal CH₃ groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles

The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Helix Stabilization Precedes Aqueous and Bilayer-Catalyzed Fiber Formation in Islet Amyloid Polypeptide

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-β-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples α-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate β-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by H solid-state NMR spectroscopy

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca²⁺-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and ²H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the ²H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of − 0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.