Selectivity in lipid binding to the bacterial outer membrane protein OmpF

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.     

Interactions of phospholipids with the potassium channel KcsA

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.     

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

The interfacial lipid binding site on the potassium channel KcsA is specific for anionic phospholipids

Lipid binding to the potassium channel KcsA from Streptomyces lividans has been studied using quenching of the fluorescence of Trp residues by brominated phospholipids. It is shown that binding of phospholipids to nonannular lipid binding sites on KcsA, located one each at the four protein-protein interfaces in the tetrameric structure, is specific for anionic phospholipids, zwitterionic phosphatidylcholine being unable to bind at the sites. The binding constant for phosphatidylglycerol of 3.0 ± 0.7 mol fraction⁻¹ means that in a membrane containing ~20 mol% phosphatidylglycerol, as in the Escherichia coli inner membrane, the nonannular sites will be ~37% occupied by phosphatidylglycerol. The binding constant for phosphatidic acid is similar to that for phosphatidylglycerol but binding constants for phosphatidylserine and cardiolipin are about double those for phosphatidylglycerol. Binding to annular sites around the circumference of the KcsA tetramer are different on the extracellular and intracellular faces of the membrane. On the extracellular face of the membrane the binding constants for anionic lipids are similar to those for phosphatidylcholine, the lack of specificity being consistent with the lack of any marked clusters of charged residues on KcsA close to the membrane on the extracellular side. In contrast, binding to annular sites on the intracellular side of the membrane shows a distinct structural specificity, with binding of phosphatidic acid and phosphatidylglycerol being stronger than binding of phosphatidylcholine, whereas binding constants for phosphatidylserine and cardiolipin are similar to that for phosphatidylcholine. It is suggested that this pattern of binding follows from the pattern of charge distribution on KcsA on the intracellular side of the membrane.     

Interaction of anilinonaphtyl labeled spectrin with fatty acids and phospholipids: a fluorescence study

Anilinonaphtyl labeled spectrin exhibits a fluorescence emission spectrum characteristic of a highly hydrophobic environment. Quenching of the fluorescence intensity by nitroxide analogs of fatty acids of affinity 10(4) M-1 reveals that the sites of interaction of fatty acids lie very close to the anilinonaphtyl groups. Similar experiments performed with a nitroxide analog of phosphatidylserine yield a 30% quenching of fluorescence while the same phosphatidylcholine analog has essentially no effect. The changes in the fluorescence emission spectrum exhibited in the presence of sonicated phosphatidylserine vesicles further outline the specificity of interaction towards phosphatidylserine, with one spectrin binding site per about 750 exposed phospholipids. Moreover, they suggest a penetration of the anilinonaphtyl group into the lipid bilayer.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase

Tryptophan residues are thought to play special roles in integral membrane proteins, anchoring transmembrane alpha-helices into the lipid bilayer. We have studied the effect of mutating the five Trp residues in the diacylglycerol kinase (DGK) of Escherichia coli to Leu residues. The fluorescence emission maxima for DGK and a variety of Trp mutants in bilayers of dioleoylphosphatidylcholine [di(C18:1)PC] are all centered at ca. 327 nm, suggesting that all five Trp residues are located close to the glycerol backbone region of the bilayer. This is also consistent with fluorescence quenching experiments, measuring the separation between the Trp residues and the bromine atoms in a bilayer of dibromostearoylphosphatidylcholine. Mutation of Trp residues in DGK was found to have significant effects on activity for DGK reconstituted into bilayers of di(C18:1)PC containing 30 mol % 1,2-dihexanoylglycerol (DHG). Of the mutants containing a single Trp residue, only that containing Trp-112 was found to give active protein. The presence of both Trp-25 and Trp-112 gave higher activity than Trp-112 alone. Trp-25 and Trp-112 are the most important Trp residues in DGK as far as activity is concerned. Effects of mutations on K(m) for DHG were generally greater than effects on v(max). The activity of wild-type and mutant DHGs reconstituted into bilayers of phosphatidylcholines was sensitive to the chain length of the phospholipid, with highest activities for chain lengths of C18 or C20 and lower activities in phosphatidylcholines with shorter or longer chains. Compared to wild-type DGK, the Trp mutants were less affected by long-chain phosphatidylcholines but more affected by short-chain phospholipids. In mutants lacking Trp-25, low activities in short-chain phospholipids followed from a decrease in v(max) compared to wild type, combined with an increase in K(m) value for DHG, as observed in the wild type. It is suggested that Trp-25 plays a role in maintaining the alignment of ATP and DHG at the active site. Fluorescence emission spectra for the Trp mutants do not change significantly with changing fatty acyl chain length from C14 to C24, showing efficient hydrophobic matching between DGK and the surrounding lipid bilayer. It is suggested that hydrophobic matching is achieved by tilting of the transmembrane alpha-helix or rotation of residues at the ends of the helices about the Calpha-Cbeta bond linking the residue to the helix backbone. As well as any structural effects, the presence of Trp residues in DGK has a clear effect on thermal stability.     

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.     

Fluorescence studies on a membrane-embedded peptide from the carboxy terminus of lipophilin

Fluorescence of an intramembranous polypeptide (T-3) derived from the carboxy-terminal sequence of lipophilin was studied in aqueous solution, detergent micelles, and lipid vesicles. In all cases, the fluorescence of the only Trp (211) was indicative of a hydrophobic, buried residue. Addition of lysophosphatidylcholine (LPC) or phosphatidylcholine (PC) gave Trp-211 a more hydrophobic, less quenching environment as compared to that in aqueous solution. Energy transfer between Trp and Tyr observed in aqueous solution was decreased by the addition of lipid or detergent. There was limited quenching by acrylamide both in the aqueous and in the lipid or detergent environments. However, PC or LPC further decreased this quenching. Cs+ and I- were even less accessible than acrylamide to Trp, further proving that the Trp was located inside the lipid bilayer. The quenching indicated that I- binds to positive charges of the protein located on the surface of the membrane. This, combined with knowledge of the sequence of lipophilin, suggested that Trp-211 was located within the membrane but was close to amino acid residues that are external to the bilayer.     

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.     

Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes

A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission lambda(max) whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.     

Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation

We have introduced single Trp residues into the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and used fluorescence quenching by brominated phospholipids to detect the presence of a binding site of high affinity for anionic phospholipids. A cluster of three positively charged residues, Arg-98, Lys-99, and Lys-100, is located on the cytoplasmic side of MscL, in a position where they could interact with the headgroup of an anionic phospholipid. Single mutations of these charged residues in the Trp-containing mutant F80W results in a decreased affinity for phosphatidic acid. Single mutations of the charged residues also result in a significant shift in the fluorescence emission spectrum in dioleoylphosphatidylcholine [di(C18:1)PC] but smaller shifts in dioleoylphosphatidic acid [di(C18:1)PA], suggesting that single mutations result in a conformational change for the protein that is reversed by interaction with anionic phospholipids. This is consistent with the observation that single mutations of the charged residues do not result in a gain of function phenotype. In contrast, simultaneous mutation of all three charged residues results in a gain of function phenotype, and a shift in fluorescence emission spectrum in di(C18:1)PC not reversed in di(C18:1)PA. The gain of function mutant F80W:V21K also shows a shifted fluorescence emission spectrum in both di(C18:1)PC and di(C18:1)PA and binds di(C18:1)PC and di(C18:1)PA with equal affinity, suggesting that the conformational change caused by the V21K mutation results in a breakup of the cluster of three positive charges. Experiments with the Trp mutants L69W and Y87W allow us to measure lipid binding constants on the periplasmic and cytoplasmic sides of the membrane, respectively. On both sides of the membrane the affinity for di(C18:1)PC is equal to that for dioleoylphosphatidylethanolamine. On the periplasmic side of the membrane, there is no selectivity for anionic phospholipids. In contrast, quenching data for Y87W provides evidence for the existence of two lipid binding sites on the cytoplasmic side of the membrane close to the Trp residue at position 87, with binding to one of these sites showing a marked preference for anionic lipid over zwitterionic lipid, presumably involving the charged cluster Arg-98, Lys-99, and Lys-100.     

Membrane interaction and cellular internalization of penetratin peptides.

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.     

Conformations of yeast alpha-mating factor and analog peptides as bound to phospholipid bilayer. Correlation of membrane-bound conformation with physiological activity

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.     

Identification of membrane-contacting loops of the catalytic domain of cytochrome P450 2C2 by tryptophan fluorescence scanning

The catalytic domain of cytochrome P450 is thought to contact the lipid core of the endoplasmic reticulum membrane based on antibody epitope accessibility, protease susceptibility, and hydrophobic surfaces present on P450 structures of solubilized forms of the proteins. Quenching by nitroxide spin label-modified phospholipids of the fluorescence of tryptophan residues substituted into cytochrome P450 2C2, modified to contain tryptophan only at position 120, was used to identify regions of P450 inserted into the lipid core and to estimate the depth of penetration. Consistent with the proposed models of cytochrome P450-membrane interaction, the fluorescence of tryptophans inserted at residues 36 and 69 in the two segments of P450 2C2 flanking the A-helix and at residue 380 in the beta2-2 strand was quenched by nitroxide spin labels on carbon 5 of the fatty acid tails of the phospholipids within the lipid bilayer. The fluorescence of tryptophan at 380 was also strongly quenched by a spin label on carbon 12 of the fatty acids suggesting it was deepest in the membrane. However, fluorescence of tryptophan substituted at residue 225 in the F-G loop, which was predicted to be in the lipid bilayer, was not quenched by the spin labels at carbons 5 and 12 of the fatty acids. The pattern of quenching of fluorescence for tryptophans at the other positions tested, 80, 189, 239, and 347, was similar to the parent protein indicating they were not inserted into the lipid bilayer as expected. The results are consistent with an orientation of cytochrome P450 2C2 in the membrane in which positions 36, 69, and 380 are inserted into the lipid bilayer and residues 80 and 225 are near or within the phospholipid headgroup region. In this orientation, the F-G loop, which contains residue 225, could form a dimerization interface as was observed in the P450 2C8 crystal structure (Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497).     

Interaction of the alpha-helices of apolipophorin III with the phospholipid acyl chains in discoidal lipoprotein particles: a fluorescence quenching study.

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding domains of apolipophorin III. The location of the Trp residues relative to the lipid bilayer was investigated in discoidal lipoprotein particles made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different single-Trp mutants of apoLp-III. A comparison of the quenching efficiencies of phospholipids containing nitroxide groups at the polar head, and at positions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interacting with the acyl chains of the phospholipid along the periphery of the bilayer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 100% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between the interaction of these two groups of helices. However, the extent of Trp fluorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesting that all Trp residues interact with the phospholipid acyl chains. This study provides the first experimental evidence of a massive interaction of the alpha-helices of apoLp-III with the phospholipid acyl chains in discoidal lipoproteins. The extent of interaction deduced is consistent with the apolipoprotein adopting a highly extended conformation.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

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.