Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.     

The interaction of human apolipoprotein C-I with sub-micellar phospholipid.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.     

The crystal structure of calcium-bound annexin Gh1 from Gossypium hirsutum and its implications for membrane binding mechanisms of plant annexins

Plant annexins show distinct differences in comparison with their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins. The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.5 A resolution and shows three metal ions coordinated in the first and fourth repeat in types II and III binding sites. Although the protein has no detectable affinity for calcium in solution, in the presence of phospholipid vesicles, we determined a stoichiometry of four calcium ions per protein molecule using isothermal titration calorimetry. Further analysis of the crystal structure showed that binding of a fourth calcium ion is structurally possible in the DE loop of the first repeat. Data from this study are in agreement with the canonical membrane binding of annexins, which is facilitated by the convex surface associating with the phospholipid bilayer by a calcium bridging mechanism. In annexin Gh1, this membrane-binding state is characterized by four calcium bridges in the I/IV module of the protein and by direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane. Analysis of the protein fold stability revealed that the presence of calcium lowers the thermal stability of plant annexins. Furthermore, an additional unfolding step was detected at lower temperatures, which can be explained by the anchoring of the N-terminal domain to the C-terminal core by two conserved hydrogen bonds.     

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.     

Calcium binding by horseradish peroxidase C and the heme environmental structure

The hyperfine shifted proton NMR spectrum of isoenzyme c of horseradish peroxidase indicated that one calcium ion is essential to the enzyme in maintaining the protein structure in the heme vicinity.     

Proton magnetic resonance spectroscopic studies of the interaction of ubiquinone-10 with phospholipid model membranes

Proton magnetic resonance spectra of ubiquinone-10 and ubiquinone-10 dispersed with dipalmitoylglycerophosphocholine or egg phosphatidylcholine in aqueous medium have been obtained. The dispersions are in the form of multilamellar liposomes as judged by 31P-NMR spectra and the thermal history of the samples have ensured that ubiquinone not incorporated into the phospholipid structure only gives rise to a broad-line NMR proton spectrum. A high-resolution proton spectrum of ubiquinone is observed with upfield shifts of the O-methyl protons of the benzoquinone rings, indicating close proximity of the molecules but with an arrangement different from the pure liquid ubiquinone. Spectra obtained in the presence of the lanthanide shift reagents, dysprosium fluorooctanedionate and Dy(NO3)3, which have a preferred location in the hydrophobic and hydrophilic domains, respectively, of ubiquinone/phospholipid codispersions, are consistent with the partitioning of ubiquinone into a hydrophobic phospholipid environment remote from the aqueous phase. The type of arrangements of ubiquinone that could be accommodated within bilayers of phospholipid are discussed.     

Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin

Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work. Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid.     

1H, 113Cd, and 31P NMR of osteocalcin (bovine gamma-carboxyglutamic acid containing protein)

The 1H (500-MHz), 113Cd (44-MHz), and 31P (81-MHz) NMR spectra of the bovine gamma-carboxyglutamate- (Gla-) containing protein osteocalcin and its Ca(II) and Cd(II) complexes in solution have been obtained. The 1H NMR spectrum of the native protein shows narrow resonances and a highly resolved multiplet structure suggesting rotational freedom of the side chains. In comparison to the simulated 1H NMR spectrum of a random polypeptide chain of the same amino acid composition, there is moderate chemical shift dispersion, indicating some conformational restraints to be present. Ca(II) binding broadens all 1H resonances, so severely at four Ca(II) ions per molecule that few structural conclusions can be made. Cd(II) substituted for Ca(II) has the same effect, and 113Cd NMR shows the Cd(II) to be in intermediate chemical exchange on the chemical shift time scale. Estimates of the chemical exchange rates required for 1H and 113Cd line broadening suggest a range of Kd values for the metal ion complexes from 10(-6) M to as high as 10(-3) M depending on the number of metal ions bound. Alternatively, 1H line broadening could be explained by relatively slow conformational fluxes in the protein induced by labile metal ion binding to one or more sites. Cd(II) when used to form a cadmium-phosphate mineral analogous to hydroxylapatite results in a crystal lattice that removes osteocalcin from solution just as effectively as hydroxylapatite. 113Cd(II) exchange at the binding sites of osteocalcin in solution is slowed dramatically by the addition of HPO4(2-). 31P NMR shows the interaction of phosphate with the protein to require the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of low-density lipoprotein with sodium deoxycholate and recombination of apoprotein B with dimyristoylphosphatidylcholine

Apoprotein B (apoB) of human plasma low-density lipoprotein (LDL) (d 1.025-1.050 g/mL) has been solubilized with solid sodium deoxycholate (NaDC) above its critical micellar concentration. ApoB is isolated by gel-filtration chromatography as a mixed micellar complex of protein and detergent in high yield in a lipid-free form. A soluble apoB-dimyristoylphosphatidylcholine (DMPC) complex has been prepared by incubation of aqueous solutions of apoB-NaDC and DMPC-NaDC (2/1 w/w) at room temperature with detergent removal by extensive dialysis. A combination of gel chromatographic and density gradient fractionation of DMPC-apoB incubation mixtures demonstrates that a reasonably well-defined complex of DMPC and apoB is formed with a 4:1 w/w lipid:protein ratio. Negative-stain electron microscopy shows these particles to be single-bilayer phospholipid vesicles with a diameter of 210 +/- 20 A into which the apoB is incorporated. Circular dichroic spectra of NaDC-solubilized apoB show apoB to have similar conformation to that seen in the native LDL particle. However, apoB that has been complexed with DMPC exhibits more alpha-helix. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band (apparent Mr 366000) for apoB after solubilization, purification, and interaction with phospholipid. The behavior of apoB during its reassociation with phospholipid and the structural features of the DMPC-apoB particle are similar to those observed in the interaction of solubilized membrane proteins with lipid rather than that of other apo-lipoproteins.     

The influence of cholesterol on the interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers

Effects of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) on thermotropic phase behavior of phosphatidylcholine/cholesterol bilayers as well as on 1H NMR spectra were studied. The surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). The benzyl group, opposite to liposomes without cholesterol, is not incorporated into the bilayer in the gel state but only in the liquid state. All the halides DBeAX (particularly the chloride DBeAC) showed greater ability to destabilize the membrane structure in the presence than in the absence of cholesterol. The interaction of DBeAX with DPPC/cholesterol bilayers and subsequent changes in the phospholipid bilayer organization depended on the kind of counterion. The strongest effects were observed for chloride (most electronegative ion) and for iodide (largest ion). The effects of chloride and bromide on phase transition and 1H NMR spectra in the presence and absence of cholesterol were opposite. This is discussed in terms of the influence of counterions on the pair cholesterol-DPPC interactions.     

Two- and three-dimensional 1H NMR studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure.

Two- and three-dimensional 1H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy proved to be a very efficient method for resonance assignments of protein 1H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by 3J alpha NH coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Structural differences in the two calcium binding sites of the porcine intestinal calcium binding protein: a multinuclear NMR study

Cadmium-113 and calcium-43 NMR spectra of Cd2+ and Ca2+ bound to the porcine intestinal calcium binding protein (ICaBP; Mr 9000) contain two resonances. The first resonance is characterized by NMR parameters resembling those found for these cations bound to proteins containing the typical helix-loop-helix calcium binding domains of parvalbumin, calmodulin, and troponin C, which are defined as EF-hands by Kretsinger [Kretsinger, R. H. (1976) Annu. Rev. Biochem. 45, 239]. The second resonance in both spectra has a unique chemical shift and is consequently assigned to the metal ion bound in the N-terminal site of ICaBP. This site is characterized by an insertion of a proline in the loop of the helix-loop-helix domain and will be called the pseudo-EF-hand site. The binding of Cd2+ to the apo form of ICaBP is sequential. The EF-hand site is filled first. Both binding sites have similar, but not identical, affinities for Ca2+: at a Ca2+ to protein ratio of 1:1, 65% of the ion is bound in the EF-hand site and 35% in the pseudo-EF-hand site. The two sites do not appear to act independently; thus, replacement of Ca2+ or Cd2+ by La3+ in the EF-hand site causes changes in the environment of the ions in the pseudo-EF-hand site. In addition, the chemical shift of Cd2+ bound to the EF-hand site is dependent on the presence or absence of Ca2+ or Cd2+ in the pseudo-EF-hand site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.