Effect of lipid unsaturation on the binding of native and a mutant form of cytochrome b5 to membranes.

The partitioning of native cytochrome b5 and a mutant form, where Trp-108 and Trp-112 were both replaced by Leu, into small unilamellar lipid vesicles was examined. The vesicles were made from phosphatidylcholines containing mono- and di-unsaturated acyl chains. As these amphipathic proteins self-associate in aqueous solution, the binding was not monitored by a simple lipid titration experiment but by an exchange assay using fluorescence quenching by brominated lipids. Each protein had a greater affinity for lipids containing mono-unsaturated chains than for vesicles containing di-unsaturated chains, and the affinities of both proteins increased in buffers of higher ionic strength. The native protein had a higher affinity than the mutant protein for all vesicles; the ratio of the affinities was relatively constant at approximately 30. This corresponds to a difference in the free energy of partitioning of 2 kcal mol(-)(1). The fluorescence quantum yields of both proteins were much lower in lipids with di-unsaturated chains whereas a similar lowering was not seen with a simple Trp compound. These data suggest that the decreased membrane hydrophobicity seen by the proteins in di-unsaturated membranes is not an inherent property of the bilayer but is induced by the insertion of the protein. Further, the similar behavior of the two proteins suggests this modulation is not sensitive to the amino acid side chains of the inserted domain.

Exchange of C(16)-ceramide between phospholipid vesicles.

Ceramide is considered to be an important signaling molecule in cellular processes such as cell growth, secretion, differentiation, and apoptosis. This implies that the molecule is able to move between cellular membranes. However, the ability of the molecule to undergo such exchange has been largely ignored despite the profound impact that this ability would have on its mechanism of action in signal-transduction cascades. With this in mind, the ability of a long-chain, radioactive ceramide, (14)C-C(16)-ceramide, to exchange between populations of lipid vesicles was evaluated. The rate of exchange of (14)C-C(16)-ceramide between lipid vesicles at lipid concentrations commonly found in cells (10-110 mM) was on the order of days (t(1/2) of 45-109 h). Simultaneous observations revealed negligible exchange of (3)H-cholesteryl oleate, which was included as a nontransferable marker to control for artifacts such as vesicle fusion and aggregation. In addition, all of the ceramide was exchangeable, and the exchange followed monoexponential kinetics, indicating that the ceramide underwent transbilayer movement at a rate faster than or equal to its rate of intervesicle exchange. Two conclusions can be drawn from these observations: (i) the spontaneous transfer of ceramide between cellular membranes is too slow to play a role in rapid, inter-membrane signaling phenomena and can only be a factor in cell functions that take place over days; and (ii) without the aid of an exchange protein, ceramide can only interact with target molecules that are located at the membrane where the ceramide is formed.

Effect of protein aggregation in the aqueous phase on the binding of membrane proteins to membranes.

Analysis of the binding of hydrophobic peptides or proteins to membranes generally assumes that the solute is monomeric in both the aqueous phase and the membrane. Simulations were performed to examine the effect of solute self-association in the aqueous phase on the binding of monomeric solute to lipid vesicles. Aggregation lowered the initial concentration of monomeric solute, which was then maintained at a relatively constant value at the expense of the aggregated solute, as the lipid concentration was increased. The resultant binding isotherm has a more linear initial portion rather than the classic hyperbolic shape. Although this shape is diagnostic of solute self-association in the aqueous phase, various combinations of values for the membrane partition coefficient and the solute self-association constant will generate similar isotherms. Data for cytochrome b5 were analyzed and, when the self-association constant was estimated by gel filtration, a unique value for the membrane partition coefficient was obtained. Thus, to obtain a true partition coefficient the state of the solute in the aqueous phase must be known. If the concentration of the monomeric solute species in the aqueous phase can be independently determined, then, even with heterogeneous aggregates, the true partition coefficient can be obtained.

Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions.

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.

Fluorescence quenching study of melittin-membrane interactions.

The bee venom peptide melittin is a popular object for studying lipid-protein interactions. In this paper we show that binding of melittin to the bilayer is a complex process involving several steps. We were able to resolve those steps by utilizing a new approach in the quantitative analysis of depth-dependent fluorescence quenching in membranes. The "distribution analysis" technique (DA) employed here provides not only the most probable depth of the fluorophore but also allows the estimation of its conformational heterogeneity and accessibility to the lipid phase. A model for melittin interaction with the membranes is suggested.

Stopped-flow fluorescence studies of the interaction of a mutant form of cytochrome b5 with lipid vesicles.

Cytochrome b5 binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.

Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties.

The structure-function relationships of the 43-amino-acid membrane-binding domain of cytochrome b5 have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in the second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and their degree of quenching by brominated lipids indicate that the mutations are not producing major conformational changes or allowing a deeper degree of penetration of the domain into the bilayer. The hydrophobicities of the three proteins were compared, by determining strengths of self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid composition, which produce no gross changes in the structure of the membrane-binding domain, will nevertheless produce very large changes in the strengths of self- and membrane-association. These differences in self-association had profound effects on the times required for membrane-association to reach equilibrium.

Fluorescence study of a temperature-induced conversion from the "loose" to the "tight" binding form of membrane-bound cytochrome b5.

Cytochrome b5 is a liver integral membrane protein that has now been expressed in, and isolated from, Escherichia coli. The structure-function relationships of the 43 amino acid membrane-binding domain (nonpolar peptide) have been examined in both native and mutant forms of the protein; in the latter, tryptophan residues at positions 108 and 112 were replaced by leucine. The temperature dependence of the fluorescence quantum yield of the Trp residues in the isolated membrane-binding domain was examined while the domain was bound to lipid vesicles. Both the lipid-bound mutant domain and lipid-bound native domain showed an irreversible increase in fluorescence above 50 degrees C. When the whole cytochrome b5 molecule, bound to lipid vesicles, was heated to this temperature, there was a conversion of the metastable, intermembrane-exchangeable ("loosely" bound), conformation to a final, virtually unexchangeable ("tightly" bound), conformation. It has been suggested previously that the protein exists in a "looped back" conformation and a "bilayer penetrating" conformation. Although the present studies are not designed to determine the absolute conformations of the loose and tight forms, the changes observed in steady-state and frequency-modulated fluorescence and the lack of change in depth of Trp 109 in the bilayer are consistent with a movement of the C-terminal segment from a looped back to a bilayer penetrating conformation as the tight form is generated.

Distribution analysis of membrane penetration of proteins by depth-dependent fluorescence quenching.

A new approach is presented to evaluate the depth-dependent quenching of the fluorescence of membrane-bound probes and integral proteins. By utilizing at least three quenchers of known and distinctly different depths, the following parameters can be recovered: most probable depth of the probe; dispersion of the depth distribution, which will depend on the size of probe and fluctuations in its position; and quenching efficiency, which is related to the exposure of a particular fluorophore to the lipid phase. The exposure of tryptophan residues in integral proteins can be quantitatively determined with respect to the model compound (tryptophan octyl ester). The proposed method was applied to the investigation of membrane complexes of the bee venom melittin and cytochrome b5.

Distribution of low density lipoprotein in the branch and non-branch regions of the aorta.

Atherosclerosis occurs focally in branch segments of the artery. Understanding why these segments are more susceptible to the development of the disease is at the root of understanding atherogenesis. We investigated accumulation of low density lipoprotein (LDL) in the branch and non-branch regions of the aorta to determine why the disease develops in branch regions. Abdominal aortas and their major branches were harvested from 36 rabbits. Rabbit LDL was prepared from whole blood and radiolabeled with 125I. The aorta was incubated with radiolabeled LDL in the lumen at 37 degrees C, under intraluminal pressure of 2-3 mmHg, for 1 h. Disks of 1.8 mm diameter were punched from the branch and non-branch regions of the aorta, cryosectioned and the sections counted in a gamma counter. Protein bound radioactivity was determined by TCA precipitation. LDL accumulation was highest towards the aortic intima and declined sharply towards the media. LDL accumulation at any given depth was higher in the branch than non-branch region. LDL accumulation in the intimal-medial sections was 87% higher in the branch than non-branch region. Total LDL accumulation in the branch was almost twice that in the non-branch region. Mean LDL accumulation was also greater in the branch than non-branch region. The aorta was significantly thicker at the branch. LDL distribution profiles indicate that LDL is present in a greater concentration and over a greater depth in the branch than non-branch region. The tendency of the branch region to accumulate LDL in greater amounts may explain its susceptibility to atherosclerotic lesion development.

Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain.

Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.

Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy.

Fourier-transform infrared spectroscopy was used to examine the secondary structure of the membrane-binding domain (nonpolar peptide) of rabbit liver cytochrome b5 in D2O and in the presence of phospholipids and deoxycholate. In all situations, the predominant structure was alpha helix, but an examination of the components of the amide I band in the spectrum of the nonpolar peptide showed that the major peak was shifted from 1655 cm-1 in the lipids to 1650 cm-1 in deoxycholate. This shift to lower frequency, together with a decrease in intensity of the amide II band, is indicative of N-H to N-D exchange of the peptide backbone. A semiquantitative analysis indicated that the alpha helix of the peptide is over 95% exchanged in the presence of deoxycholate but is only 10% exchanged in the presence of lipid. These data suggest that the membrane-inserted portion of the peptide is alpha helical and is largely protected from N-H to N-D exchange by the bilayer. We suggest that this technique appears to provide a general method for determining the type of secondary structure involved in membrane interaction and the percentage of this structure which is involved in the interaction.

Quenching of tryptophan fluorescence by brominated phospholipid.

Bromolipids [1-palmitoyl-2-(dibromostearoyl)phosphatidylcholine] with bromines at the 4,5-, 6,7-, 9,10-, 11,12-, and 15,16-positions were used to examine the fluorescence quenching of a synthetic, membrane-spanning peptide (Lys2-Gly-Leu8-Trp-Leu8-Lys-Ala-amide) incorporated into both small and large unilamellar vesicles. The peptide-lipid vesicles were analyzed to show that at least 75% of the peptide was in a transbilayer configuration, placing the single tryptophan in its predicted place in the center of the bilayer. Quenching profiles of the peptide in bromolipid showed maximal (90%) quenching by the 15,16-bromolipid, indicating that the bromolipids can accurately locate the position of a tryptophan in the bilayer. The quenching by the other bromolipids decreased with an r6 dependence and an apparent R0 of 9 A. In addition, indole in methanolic solution was subjected to quenching by a variety of mono- and dibrominated hydrocarbons. The quenching was analyzed, by using a modified Stern-Volmer equation, and found to be greatly dependent upon the number and positioning of the bromines. Monobromobutanes were found to have a quenching efficiency of only 7% while dibromobutanes, with bromines on adjacent carbon atoms, had efficiencies of over 80%. In addition, the dibromobutanes exhibited significant "static" quenching whereas the monobrominated butanes did not. These data suggest that the bromolipids are more appropriately defined as short-range quenchers rather than strictly contact quenchers.

Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy.

Fourier-transform infrared spectroscopy was used to examine the secondary structure of rabbit liver cytochrome b5 and the polar and nonpolar domains of the protein. The data for both the polar and nonpolar domains agree well with those previously obtained by other physical techniques. In particular it was found that the nonpolar membrane-binding domain was predominantly alpha helix and that the polar domain was also highly helical, but not all alpha helix. The independence of the two domains in the whole molecule was, in general, confirmed by the additivity of the spectra of the two domains. The small differences that were seen indicate that there is a loss of alpha helix when the protein is cut into the two domains. In addition, there appeared to be a slight difference in the exposure to solvent of the amide NH groups in the alpha-helical portion of the nonpolar domain when it was examined in isolation.

Infrared spectroscopic analysis of salt bridge formation between cytochrome b5 and cytochrome c.

The infrared spectrum of a solution of a protein contains bands due to both the peptide backbone and the amino acid side chains. Generally, the bands due to the peptide backbone, between 1700 and 1600 cm-1, are analyzed to determine the secondary structure of the protein; the bands due to the amino acid side chains, between 1600 and 1500 cm-1, are largely ignored. When cytochrome b5 is mixed with cytochrome c, under conditions that favor ionic complex formation, changes are seen in protein secondary structure and also in a band at 1562 cm-1. The band at 1562 cm-1 is due to the side-chain carboxyl of Glu residues, rather than those of Asp residues that show a band at 1585 cm-1, and the changes in the band at 1562 cm-1 indicate that when the two proteins interact, three ionized carboxyl groups of Glu become involved in salt bridge formation. This result is identical with that obtained by previous theoretical studies and suggests that infrared spectroscopy may be a rapid and quantitative method for the study of ionic interactions between proteins.

Determination of the depth of bromine atoms in bilayers formed from bromolipid probes.

X-ray diffraction analysis has been performed on a series of 1-palmitoyl-2-dibromostearoyl-phosphatidylcholines (BRPCs) with bromine atoms at the 6, 7-, the 11, 12-, or the 15, 16-positions on the sn-2 acyl chains. The diffraction patterns indicate that, when hydrated, each of these lipids forms liquid-crystalline bilayers at 20 degrees C. For each lipid, electron density profiles and continuous Fourier transforms were calculated by the use of swelling experiments. In the electron profiles, high-density peaks, due to the bromine atoms, are observed. The separation between these bromine peaks in the profile decreases as the bromine atoms are moved toward the terminal methyl of the acyl chain. For the 6, 7- and 11, 12-bromolipids, experimental Fourier transforms can be approximated by the sum of the transform of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and the transform of two symmetrically placed peaks of electron density (the bromines). For the case of the 15, 16-bromolipids, a better fit is obtained for the transforms of a model bilayer where the thickness of the methylene chain region of the bilayer is 3 A greater than that of POPC. Our analysis indicates the following: for each of these bromolipids, the bromines are well localized in the bilayer; the distance of the bromines from the head-group-hydrocarbon boundary are 3.5, 8.0, and 14 A, for 6, 7-, 11, 12-, and 15, 16-BRPC, respectively; the bilayer thickness and perturbation to bilayer hydrocarbon chain packing caused by the bromine atoms depend on the position of the bromines on the hydrocarbon chain.

Fluorescence studies of cytochrome b5 topography. Incorporation of cytochrome b5 into brominated phosphatidylcholine vesicles by deoxycholate.

Cytochrome b5 was incorporated into large vesicles of 1-palmitoyl-2-dibromostearoylphosphatidylcholine by mixing lipid, protein, and deoxycholate followed by removal of the detergent by gel filtration. The tryptophan fluorescence emanating from the hydrophobic membrane-binding domain was quenched more effectively when the bromine atoms were in the 6,7-positions than when they were in the 15,16-positions of the acyl chain. To more precisely define the position of the quenchable tryptophan, the experiment was repeated with lipids with the bromine atoms at the 4,5-, 6,7- or 9,10-positions. Again the 6,7 species was the most efficient quencher. The cytochrome b5 bound to these vesicles would not transfer to small unilamellar sonicated vesicles and so was in the "tight" configuration. If the cytochrome were added to the vesicles after the detergent was removed, the same order of quenching was seen but the cytochrome would transfer to other vesicles. These data indicate that the quenching of the tryptophan fluorescence is greatest when the bromines are at the 6,7-positions whether the vesicles are large or small and whether the cytochrome is in the tight or "loose" configuration and so place the tryptophan 0.7 nm below the vesicle surface in all of these membranes.

Fluorescence quenching of cytochrome b5 in vesicles with an asymmetric transbilayer distribution of brominated phosphatidylcholine.

Several fluorescence techniques have been used to estimate the depth, in the membrane, of the endogenous tryptophans of membrane-bound proteins. We reported recently the use of phosphatidylcholines specifically brominated at different positions of the sn-2 acyl chain for this purpose (Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. (1985) Biochemistry 24, 2895-2901). The membranes made from these brominated lipids will have the brominated lipid in both monolayers, and so the estimated depth of the fluorophore will be relative to either the inner or outer surface of the membrane, but will not distinguish between these two extremes. To differentiate between these two models vesicles have now been made with an asymmetric distribution of brominated lipid, by use of phosphatidylcholine exchange protein. The asymmetric vesicles were isolated by virtue of their density, and their asymmetry was established by addition of an amphipathic fluorescent carbazole compound. With these vesicles it was shown that the tryptophan in the membrane-binding domain of cytochrome b5 which is quenched by bromolipid is located 0.7 nm below the outer surface of the membrane vesicles, rather than 0.7 nm from the inner surface.