Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used ³¹P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and ³¹P T₂ relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T₂ relaxation are observed at higher temperatures. A comparison of ³¹P T₁ values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T₁ relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules  – in particular for the negatively charged DMPG – while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide–membrane interactions.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Diffusion and dynamics of penetratin in different membrane mimicking media

The interaction between the cell-penetrating peptide (CPP) penetratin and different membrane mimetic environments has been investigated by two different NMR methods: ¹⁵N spin relaxation and translational diffusion. Diffusion coefficients were measured for penetratin in neutral and in negatively charged bicelles of different size, in sodium dodecyl sulfate micelles (SDS), and in aqueous solution. The diffusion coefficients were used to estimate the amount of free and bicelle/micelle-bound penetratin and the results revealed that penetratin binds almost fully to all studied membrane mimetics. ¹⁵N relaxation data for three sites in penetratin were interpreted with the model-free approach to obtain overall and local dynamics. Overall correlation times for penetratin were in agreement with findings for other peptides of similar size in the same solvents. Large differences in order parameters were observed for penetratin in the different membrane mimetics. Negatively charged surfaces were seen to restrict motional flexibility, while a more neutral membrane mimetic did not. This indicates that although the peptide binds to both bicelles and SDS micelles, the interaction between penetratin and the various membrane mimetics is different.     

Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

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.     

Morphology of fast-tumbling bicelles: a small angle neutron scattering and NMR study

Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures.     

NMR solution structure and dynamics of motilin in isotropic phospholipid bicellar solution

The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues, have been studied in the presence of isotropic q=0.5 phospholipid bicelles. The NMR solution structure of the peptide in acidic bicelle solution was determined from 203 NOE-derived distance constraints and six backbone torsion angle constraints. Dynamic properties for the ¹³C-¹H vector in Leu10 were determined for motilin specifically labeled with ¹³C at this position by analysis of multiple-field relaxation data. The structure reveals an ordered -helical conformation between Glu9 and Lys20. The N-terminus is also well structured with a turn resembling that of a classical -turn. The ¹³C dynamics clearly show that motilin tumbles slowly in solution, with a correlation time characteristic of a large object. It was also found that motilin has a large degree of local flexibility as compared with what has previously been reported in SDS micelles. The results show that motilin interacts with the bicelle, displaying motional properties of a peptide bound to a membrane. In comparison, motilin in neutral bicelles seems less structured and more flexible. This study shows that the small isotropic bicelles are well suited for use as membrane-mimetic for structural as well as dynamical investigations of membrane-bound peptides by high-resolution NMR.     

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.     

Calcium Enhances Binding of Aβ Monomer to DMPC Lipid Bilayer

Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aβ monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aβ monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp²³-Lys²⁸. Second, calcium strengthens Aβ peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aβ monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aβ monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aβ binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aβ-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aβ peptide; and 2) destabilization of the Asp²³-Lys²⁸ salt bridge makes Lys²⁸ available for interactions with the bilayer. Finally, we conclude that a single Aβ monomer does not promote permeation of calcium ions through the zwitterionic bilayer.     

High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium

A mixture of dihexanoyl phosphatidylcholine and dimyristoylphosphatidylcholine in water forms disc-shaped particles, often referred toas bicelles [Sanders and Schwonek (1992) Biochemistry, 31, 8898–8905].These adopt an ordered, liquid crystalline phase, which can be maintained atvery low concentrations of the bicelles (down to 3% w/v). At thisconcentration the spacing between individual bicelles, on average, exceeds300 Å. The bicelles are shown to have a negligible effect on therotational diffusion of ubiquitin as judged by the ¹⁵NT₁ values of the backbone amides relative to those inisotropic aqueous solution. The protein exhibits a residual degree ofalignment which is proportional to the bicelle concentration, andapproximately collinear with ubiquitin's rotational diffusion tensor. Thedegree of alignment obtained offers unique opportunities for studying theprotein's structure and dynamics.     

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS)

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by ³¹P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7°, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0° toward 54.7°. Thus the relative sign of dipolar and scalar couplings can be determined.     

A comparative study of the effect of cholesterol on bicelle model membranes using X-band and Q-band EPR spectroscopy

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (S_mol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.     

Assessing the size, stability, and utility of isotropically tumbling bicelle systems for structural biology

Aqueous phospholipid mixtures that form bilayered micelles (bicelles) have gained wide use by molecular biophysicists during the past 20 years for spectroscopic studies of membrane-bound peptides and structural refinement of soluble protein structures. Nonetheless, the utility of bicelle systems may be compromised by considerations of cost, chemical stability, and preservation of the bicelle aggregate organization under a broad range of temperature, concentration, pH, and ionic strength conditions. In the current work, ³¹P nuclear magnetic resonance (NMR) and atomic force microscopy (AFM) have been used to monitor the size and morphology of isotropically tumbling small bicelles formed by mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine (DIOMPC) with either 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-di-O-hexyl-sn-glycero-3-phosphocholine (DIOHPC), testing their tolerance of variations in commonly used experimental conditions. ¹H-¹⁵N 2D NMR has been used to demonstrate the usefulness of the robust DMPC-DIOHPC system for conformational studies of a fatty acid-binding protein that shuttles small ligands to and from biological membranes.     

Motilin-bicelle interactions: membrane position and translational diffusion

The interaction between the peptide hormone motilin and bicelles has been investigated by pulsed field gradient-nuclear magnetic resonance methods and by the use of paramagnetic probes. Diffusion coefficients were measured for motilin, the phospholipids with and without motilin, and for tetramethylsilane. The results show that around 90% of motilin is bound to acidic bicelles and 84% of motilin is bound to neutral bicelles. It is found that the apparent bicelle size is reduced by the presence of motilin. This cannot be explained by changes in 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine solubility. The use of paramagnetic agents to investigate the position of motilin shows that the turn in the N-terminus of motilin is inserted into the bicelle, while the helix most likely resides within the head-group layer.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Cation modulation of bicelle size and magnetic alignment as revealed by solid-state NMR and electron microscopy

The influence of salts (KCl, NaCl, CaCl(2), and MgCl(2)) on bicelles (bilayered micelles) made of dimyristoylphosphatidylcholine (DMPC, molar fraction X = 78%) and dicaproylphosphatidylcholine (DCPC) was investigated by solid-state (31)P- and (2)H NMR as well as by freeze-fracture electron microscopy. Sizes were determined from (2)H- and (31)P NMR on the basis of a model that incorporated a planar bilayer and a (half-torus) curved rim representing the DMPC and DCPC regions of the bicelle, respectively. Good agreement was shown with sizes determined independently from freeze-fracture electron microscopy images. In the presence of K(+) and Na(+), bicelles have diameters of approximately 300 A while in the presence of Ca(2+) and Mg(2+); their diameter increases to approximately 500 A. Bicelle magnetic alignment is considerably improved by the presence of salts. The optimum salt concentration for such an effect ranges from 50 to 200 mM. Bicelles are magnetically aligned for temperatures roughly ranging from 30 degrees C to 40 degrees C with monovalent cations; this range is slightly extended in the presence of divalent salts. In this temperature range, the dynamics of the long-chain hydrocarbon region of the bicelle (leading to a bicelle thickness of 38 A) and of water is about the same independently of cation nature and concentration. However, at higher temperatures, considerable differences in water dynamics are observed between systems with monovalent and divalent cations. In these conditions, the system consists of a mixture of micelles and extended bilayers, which show residual macroscopic alignment in the magnetic field.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Mechanism of penetration of Antp(43-58) into membrane bilayers

Antp(43-58) is one of many peptides with basic and aromatic residues capable of crossing cell membranes efficiently in a receptor-independent manner. The basic-aromatic motif is responsible for peptide binding to the negatively charged surface of membrane bilayers. However, the mechanism of membrane penetration is unclear. We use high-resolution (1)H solution NMR methods to establish the location of the Antp(43-58) peptide bound to membrane bicelles composed of DMPC, DMPG, and DHPC, and compare it to the location of an Antp(43-58) variant which is not able to cross cell membranes. Two critical tryptophans are substituted with phenylalanine in this variant (W48F and W56F). Additional (31)P and (2)H NMR measurements of membrane bicelles are used to probe the changes in orientation of the lipid headgroups and the changes in the mobility or segmental order of the lipid acyl chains upon peptide binding. We find that Trp48 and Trp56 of Antp(43-58) insert into the hydrophobic core of the membrane and that this induces a change in the orientation of the negatively charged DMPG headgroups. The depth of insertion and the change in lipid orientation are concentration-dependent and argue for an electroporation-like mechanism for membrane penetration.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.