©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Peptides Corresponding to Fatty Acylation Sites in Proteins with Model Membranes (*)

Mercy Joseph , Ramakrishanan Nagaraj (§)

From the (1)Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In recent years, a large number of proteins having covalently linked myristic and palmitic acids have been discovered. It is assumed that fatty acid acylation serves to anchor proteins to membranes. However, it is not clear whether fatty acids modulate orientation of peptide chain in membranes or help in associating hydrophilic segments of peptides with membranes. We have examined the aggregation properties and membrane association of peptides corresponding to myristoylation and palmitoylation regions of proteins by fluorescence spectroscopy. Both acylated and non-acylated peptides were used for investigation. Binding of the peptides to lipid vesicles was assessed by monitoring the fluorescence of tryptophan as well as the quenching of its fluorescence in the presence of quenchers like I and acrylamide. Our results indicate that in the peptide corresponding to a transmembrane segment, palmitoylation results in a change in the orientation of the peptide chain in the lipid bilayer. In the case of peptides that do not have a hydrophobic segment, acylation with palmitic or myristic acid does not appear to result in increased binding to lipid bilayer. Our results suggest that (i) the primary role of myristoylation may not be an anchor for membrane attachment as assumed, (ii) palmitoylation in the case of proteins having transmembrane segments may serve to realign the transmembrane segment from the normal orientation perpendicular to the bilayer surface, (iii) in the case of proteins where there is no hydrophobic segment, palmitoylation may not serve as a membrane anchor and could be involved in interaction with other membrane-bound proteins.


INTRODUCTION

A large number of proteins undergo chemically diverse modifications during or after their translation (Wold, 1981). These modifications have an effect on their structures and functions (Wold, 1981). One modification that has attracted considerable attention over the years is acylation with myristic and palmitic acids (Schultz et al., 1988; Schmidt, 1989) and isoprene units (Sinensky and Lutz, 1992). There has been extensive documentation of proteins that are acylated and the amino acid sequences in the region of acylation (Schultz et al., 1988; Schmidt, 1989). Mutagenesis studies have shown that attachment of fatty acid to protein is important for function of the proteins (Kamps et al., 1985; Willumsen et al., 1984). Enzymes that are responsible for attachment of fatty acids to proteins have been identified (Berger and Schmidt, 1987; Towler et al., 1987; Glover et al., 1988). It appears that myristoylation is cotranslational (Wilcox et al., 1987) and palmitoylation is posttranslational (Schultz et al., 1988). A large number of fatty acylated proteins are either growth factor receptors (James and Olson, 1990) or participate in signal transduction (James and Olson, 1990) and are located at the vicinity of membranes. Hence, it has been postulated that fatty acid acylation serves to anchor proteins to membranes. However, there is practically no experimental evidence indicating whether fatty acids modulate orientation of peptide chains in membranes or help in associating hydrophilic segments of peptides with membranes. Hence, it would be pertinent to study the interaction between lipids and peptides corresponding to acylation sites in proteins. An understanding of the biophysical nature of the interactions of fatty acylated peptides with model membranes will help in understanding the molecular properties involved in the association of fatty acylated peptides with membranes in cells.

In the present study, we have examined the aggregation properties and membrane association of peptides corresponding to myristoylation and palmitoylation regions of proteins (). Both acylated and non-acylated peptides were used for investigations. The peptides chosen correspond to acylated regions that are hydrophobic as well as hydrophilic in nature. All the peptides have a tryptophan residue. Hence, peptide association and lipid-peptide interactions were assayed by steady state fluorescence measurements of tryptophan. Changes in fluorescence emission intensity and in the wavelength of emission maximum that occur when tryptophan enters an environment with a smaller dielectric constant (Creed, 1984) served as an indication of peptide aggregation or association with lipid. In addition to these measurements, quenching experiments in which the exposure of the peptides to aqueous quenchers like I (Lehrer, 1971) and membrane-penetrating quenchers like acrylamide (Eftink and Ghiron, 1976) were determined. The combined approach of applying intrinsic fluorescence and quenching measurements provided data that indicate how the peptides aggregate and associate with model membranes. Based on the results, the possible role of fatty acid acylation in proteins has been discussed.


EXPERIMENTAL PROCEDURES

Materials

Amino acids, dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, fatty acids, and ethane dithiol were purchased from Sigma; solvents were from Merck India; trifluoroacetic acid, thioanisole, and m-cresol were from Fluka. All Fmoc()-protected amino acids were from Nova Biochem.

Methods

Peptide Synthesis

All peptides were synthesized by solid phase procedures. Peptide 31R () was synthesized using 2% hexane dioldiacrylate-cross-linked polystyrene resin (anchoring capacity of 2.32 meq/g of resin) by procedures described earlier (Renil et al., 1994). Synthesis of peptides 12R, 11R, and 14R () were performed on a Pharmacia semi-automated synthesizer by Fmoc chemistry (Atherton and Sheppard, 1989) using NovaSyn KA resin from Novabiochem. The procedure for acylation of cysteine with palmitoyl chloride and of the N-terminal glycine with myristoyl chloride, when attached to the resin after removal of the acetamido methyl group was achieved as described previously (Joseph and Nagaraj, 1993). This was followed by acidolytic cleavage of the peptide from the resin to generate the fatty acylated peptides 31Pal, 12Pal, 11Myr, and 14Myr.

The peptides were purified by fast performance liquid chromatography on a reverse phase PepRPC 5/5 (Pharmacia) column, except peptides 31R and 31Pal. Peptides 31R and 31Pal were purified by thorough washing with MeOH and acetonitrile as the impurities dissolved in small volumes of these solvents. The purity of the peptides were determined by amino acid analyses on a LKB 4151 Apha Plus Amino Acid Analyzer, after hydrolysis in vacuo with trifluoroacetic acid, 6 N HCl (1:2). The presence of fatty acids was confirmed by analysis on a Hewlett Packard 5840A gas-liquid chromatograph, after hydrolysis. The purity of non-acylated peptides was further confirmed by sequencing on a model 473A protein sequencer (Applied Biosystems). Peptide stock solutions were made in methanol and/or MeSO, and the concentrations were estimated by quantitative amino acid analysis.

Preparation of Vesicles

Small unilamellar vesicles were prepared by sonication of aqueous suspension of purified egg phosphatidylcholine (PC) in an ice bath with a 0.5-cm flat disrupter tip mounted on a Branson sonifier (cell disruptor B30 manufactured by the Branson Sonic Power Co.) and with a power setting at 2. After sonication, the vesicle suspension was centrifuged to remove titanium particles.

Fluorescence Measurements

Steady state fluorescence measurements were performed with a Hitachi F4010 spectrofluorometer, using 1-cm pathlength quartz cuvettes. Excitation and emission bandpass was set at 3 nm each.

Binding of Peptides to Lipid Vesicles

The binding of peptides to lipid vesicles was assessed by monitoring the changes in the tryptophan spectra of the peptides on addition of small unilamellar vesicles of egg PC. Small aliquots of vesicle suspension were added to a solution of 3 µM peptide in buffer (5 mM Hepes, pH 7.4). The suspension was continuously stirred; after each addition of lipid, it was left to equilibrate for 20 min before the fluorescence spectra were recorded. Fluorescence intensities were corrected for contribution of light scattering caused by the lipid vesicles.

Quenching of Fluorescence by Acrylamide and Iodide

The peptides were taken in 1 ml of buffer (5 mM Hepes, pH 7.4). The excitation wavelength used was 295 nm for acrylamide quenching and 280 nm for iodide quenching, respectively. The excitation and emission bandpass were set at 5 nm each, and the emission was checked at 340 nm. Increasing aliquots of an 3 M solution of acrylamide were added to the continuously stirred peptide solution and to the lipid vesicles containing peptide. (The peptide concentration was 3 µM, and lipid concentration was 150 µM). Fluorescence intensities were corrected for dilution effects. Aliquots of KI were added from a 4 M stock solution of KI containing 1 mM of NaSO in order to prevent formation of I and I, to the peptide and peptide-lipid solutions. The fluorescence intensity was measured at 340 nm, and the values were corrected for dilution effects. The quenching data were analyzed according to the Stern-Volmer equation. The fluorescence measurements were done on a Hitachi F4010 spectrofluorometer using a 1-cm pathlength quartz cuvette. All measurements were done three times, and the average value was taken for analysis.

Carboxyfluorescein (CF) Assay

The ability of peptides to cause perturbation of lipid vesicles was monitored by noting the increase in fluorescence intensity of carboxyfluorescein (Weinstein et al., 1977; Blumenthal et al., 1977) encapsulated in PC at high self-quench concentrations, on addition of peptides The initial value was taken as zero, and after the addition of peptide to the lipid vesicles the release of CF was monitored continuously as a function of time and as a function of peptide concentration. The fluorescence intensity obtained on addition of 0.1% Triton X-100 was taken as 100%. No discernible CF release was obtained with vesicles alone during the time course of the experiment.

Polarization

Polarization measurements were done with peptides in buffer and in lipid vesicles (peptide concentration was 3 µM and lipid concentration was 150 µM) using a Hitachi polarization accessory. Polarization values were calculated from the formula shown in Equation 1, where I and I are the measured fluorescence intensities with the excitation polarizer vertically oriented and the emission polarizer vertically and horizontally oriented, respectively. G is the correction factor and is equal to I/I (Chen and Bowman, 1965).

On-line formulae not verified for accuracy


RESULTS

Aggregation of Peptides

Since the peptides listed in are hydrophobic either due to the peptide sequence or attached fatty acid, they could aggregate in aqueous medium. Hence this aspect was examined first by monitoring the of Trp. The of Trp is sensitive to solvent polarity (Lakowicz, 1983). Emission maximum of 350 nm is indicative of Trp exposure to aqueous environment, whereas values of 340 and 330 nm indicate moderate exposure to and shielding from aqueous environment respectively.

The emission spectra of the peptides 12R and 31R and their palmitoylated derivatives are shown in Fig. 1. The emission maximum is 350 nm for 12R and 12Pal, 330 nm for 31R, and 345 nm for 31Pal. The position of the emission maximum indicates that the tryptophan residue is exposed to aqueous environment in 12R and 12Pal. The emission maximum of 31R indicates that the tryptophan residue is shielded from aqueous environment. However, in 31Pal, the position of the emission maximum (345 nm) indicates that the tryptophan is moderately exposed to the aqueous environment. The emission spectra of 11R, 14R, and their myristoylated derivatives are presented in Fig. 2. The emission maxima (350 nm) indicate that the tryptophan is completely exposed in 11R as well as in 11Myr, whereas it is moderately shielded from the aqueous environment in 14R and 14Myr ( 340 nm).


Figure 1: Fluorescence emission spectra of peptides in buffer (5 mM Hepes, pH 7.4). a, 12R (--) and 12Pal (- - -); b, 31R (--) and 31Pal (- - -). Excitation wavelength was at 280 nm. The peptide concentration was 3 µM.




Figure 2: Fluorescence emission spectra of peptides in buffer (5 mM Hepes, pH 7.4). a, 11R (--) and 11Myr (- - -); b, 14R (--) and 14Myr (- - -). Excitation wavelength was at 280 nm. The peptide concentration was 3 µM.



The fluorescence spectra were then recorded as a function of peptide concentration (results not shown). For all the peptides except 14R and 14Myr a linear increase in fluorescence intensity is observed. In addition, there is no shift in the position of emission maximum or hysteresis, which are indicative of peptide aggregation (Mathew et al., 1980). Thus, the peptides 12R, 12Pal, 11R, and 11Myr do not aggregate in the range of 1-6 µM. A slight non-linearity in the appearance of the curves was observed in the case of 31R, 31Pal, 14R, and 14Myr, suggesting a tendency to aggregate in buffer. It is also conceivable that 31R and 14R are already aggregated at a concentration of 1 µM.

Since denaturants like guanidine hydrochloride and urea (De Young et al., 1993) and solvents like methanol could conceivably influence the aggregation properties of these peptides, the emission spectra of 31R, 14R, and their acylated derivatives were examined in these media. The spectra of these peptides in urea, guanidine hydrochloride, and MeOH are shown in Fig. 3. In 31R, the emission maximum does not shift in the presence of denaturants but there is an increase in quantum yield. This suggests that even in an unfolded state the tryptophan residue is not exposed to aqueous environment. However, a red shift in emission maximum and increase in quantum yield indicates ``unfolding'' of the peptide, resulting in exposure of Trp to aqueous environment. In 31Pal, there is very little shift in emission maximum or change in quantum yield. In 14R, although there is an increase in quantum yield, there is no shift in the position of emission maximum and 14Myr peptide does not exhibit any spectral change. It thus appears that in the hydrophobic peptides, the tryptophan residue seems to be solvent-shielded even under conditions where these peptides would be unfolded and disaggregated. The emission maxima and relative intensities of fluorescence of these peptides in different environments are summarized in . These correspond to an average value of three independent sets of experiments. The variation in values was 4%.


Figure 3: Fluorescence emission spectra of peptides in presence of denaturants. a, 31R, b, 31Pal, c, 14R, d, 14Myr in urea (--), guanidine hydrochloride (- - -), and methanol (---). Excitation wavelength was at 280 nm. Peptide concentration was 3 µM, urea concentration was 6 M, and guanidine hydrochloride concentration was 8 M.



Binding to Lipid Vesicles

The spectra of the peptides in the presence of small unilamellar vesicles of egg PC are shown in Fig. 4. In the case of hydrophilic peptides and their acyl derivatives, there is a decrease in quantum yield in the presence of lipid vesicles. The hydrophobic peptide 31R does not show much decrease in fluorescence intensity on association with lipid vesicles but in the palmitoylated derivative 31Pal, there is considerable decrease in quantum yield. In the case of 14R, a 14-residue leucine-rich peptide, there is an increase in quantum yield, but in the myristoylated derivative of 14R, the increase in relative fluorescence intensity is almost double that of 14R. In order to ascertain that the peptides did indeed bind to lipid vesicles, the fluorescence behavior of the peptides at a fixed concentration as a function of lipid concentration was determined. The results are presented in Fig. 5. A continuous decrease in fluorescence is observed for 12R, 11R, and their acyl derivatives. While there seems to be leveling of fluorescence in the case of 12R and 12Pal peptides, it is not apparent with 11R and 11Myr peptides. 31R shows very little change in fluorescence with increasing concentration of lipid vesicles, whereas 31Pal shows a sharp decrease, which levels off at a lipid:peptide ratio of 50:1. In the case of 14R and 14Myr peptides, there is an initial increase in fluorescence intensity, which decreases with increasing concentrations of lipid. The data shown in Fig. 5suggest that 12R, 11R, and their acyl derivatives bind to lipid vesicles although not very strongly. It is unlikely that 31R does not bind to lipid vesicles. Since the emission maximum of 335 nm indicates a hydrophobic environment, there may be no further change in the environment of tryptophan on binding to lipid vesicles. The acylated peptide 31Pal clearly binds to lipid vesicles.


Figure 4: Comparison of fluorescence emission spectra of peptides in buffer (5 mM Hepes, pH 7.4) and lipid vesicles of egg PC. a, 12R in buffer (--) and lipids (---), 12Pal in buffer (- - -) and lipids (--). b, 11R in buffer (--) and lipids (---), 11Myr in buffer (- - -) and lipids (--). c, 31R in buffer (--) and lipids (---), 31Pal in buffer (- - -) and lipids (--). d, 14R in buffer (--) and lipids (---), 14Myr in buffer (- - -) and lipids (--). Excitation wavelength was at 280 nm. The peptide concentration was 3 µM, and lipid concentration was 150 µM.




Figure 5: Fluorescence value of the peptides at emission maximum as a function of lipid concentration in aqueous buffer. Peptide concentration was kept at 3 µM. Excitation wavelength was at 280 nm.



It is unlikely that the decrease in quantum yield is due to impurities present in lipid vesicles, as similar results were obtained with dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine. Additionally, peptides SPF and SPLN, the fluorescence behavior of which has been well established (Sitaram and Nagaraj, 1993), showed an increase in quantum yield in lipid preparations used in the present study, indicating that the quenching observed is a real observation.

Fluorescence quenching experiments were undertaken to map the accessibility of the tryptophan residues to soluble and membrane resident quenchers. The quenching data by iodide analyzed by Stern-Volmer plots are shown in Fig. 6. The Stern-Volmer quenching constants are very similar for 12R, 11R, and their acyl derivatives in the presence and absence of lipid vesicles indicating that the accessibility of Trp to I in the two situations is quite similar. It thus appears that the Trp in these peptides is not located in the hydrophobic core but is exposed to aqueous environment. In the case of 31R, the Trp is not accessible either in buffer or in lipid vesicles, whereas in the palmitoylated peptide, the Trp is not accessible in lipid vesicles and accessible in buffer. The quenching behavior of Trp in 14R is very similar to that of 31R. The iodide quenching data clearly indicate that 14Myr and 31Pal associate with lipid vesicles. The evidence for 11Myr and 12Pal association with lipid vesicles is less compelling. The quenching data analysis by Stern-Volmer plot in presence of acrylamide is shown in Fig. 7. The data support the conclusion drawn on the basis of KI quenching experiments. Interestingly, the efficient quenching observed in 31R reiterates the very hydrophobic environment in which the Trp is situated.


Figure 6: Stern-Volmer plots for quenching Trp fluorophore of peptides in buffer and lipid vesicles by KI. a, 12R in buffer () and lipids (), 12Pal in buffer () and lipids (). b, 11R in buffer () and lipids (), 11Myr in buffer () and lipids (). c, 31R in buffer () and lipids (), 31Pal in buffer () and lipids (). d, 14R in buffer () and lipids (), 14Myr in buffer () and lipids (). The peptide concentration was 3 µM, and lipid concentration was 150 µM. Excitation and emission wavelengths were set at 280 and 340 nm.




Figure 7: Stern-Volmer plots for quenching Trp fluorophore of peptides in buffer and lipid vesicles by acrylamide. a, 12R in buffer () and lipids (), 12Pal in buffer () and lipids (). b, 11R in buffer () and lipids (), 11Myr in buffer () and lipids (). c, 31R in buffer () and lipids (), 31Pal in buffer () and lipids (). d, 14R in buffer () and lipids (), 14Myr in buffer () and lipids (). The peptide concentration was 3 µM, and lipid concentration was 150 µM. Excitation and emission wavelengths were at 295 and 340 nm.



The fluorescence polarization of Trp in the peptides in buffer and lipid vesicles are summarized in I. In the presence of lipid vesicles, there is an increase in polarization for all the peptides. The polarization values indicate a greater degree of immobilization in the case of acylated hydrophobic peptides. The similar polarization values observed in the hydrophilic peptides suggest that acylation does not lead to immobilization of Trp in lipid vesicles. The environment and flexibility of tryptophan appears to be very similar in the acylated and nonacylated hydrophilic peptides.

In order to examine whether association of the peptides with lipid vesicles results in perturbation of the bilayer structure, the release of CF from lipid vesicles entrapped at self-quench concentrations was monitored. The data presented in Fig. 8indicate that the association of the peptide 14R and its acyl derivative with PC vesicles results in CF efflux, indicating the perturbation of the bilayer. The myristoyl derivative 14Myr clearly permeabilizes the vesicles to a greater extent than the peptide without the fatty acid.


Figure 8: Release of encapsulated carboxyfluorescein from PC vesicles in presence of peptides 14R (A), 14Myr (B), and other peptides (C). Small unilamellar vesicles (150 µM) containing trapped CF were suspended in 1 ml of Hepes buffer (5 mM Hepes, pH 7.4, 100 mM NaCl). Peptides were added at t = 0. Increase in fluorescence was monitored as a function of time. Initial fluorescence is taken as zero, and the fluorescence on addition of Triton X-100 (indicated by ) is taken as maximum. Excitation and emission wavelengths were at 493 and 520 nm. a, 14R at peptide:lipid = 2:50. b, 14R at peptide:lipid = 1:150. c, 14R at peptide:lipid = 0.5:150. d, 14Myr at peptide:lipid = 1.5:150. e, 14Myr at peptide:lipid = 1:150. f, 14Myr at peptide:lipid = 0.5:150. g, 31R, 31Pal, 12R, and 12Pal at peptide:lipid = 1:150. h, buffer + lipid.




DISCUSSION

The palmitoylation site in proteins having transmembrane-spanning segments is in the vicinity of a hydrophobic stretch of amino acids (Grand, 1989). In other proteins like Ras, the palmitoylation site is not in the vicinity of a hydrophobic stretch (Hancock et al., 1989). The myristoylation site is at the N terminus of proteins and is generally not in a hydrophobic region (Schultz et al., 1988). The peptides (listed in ) used in this study correspond to the palmitoyl and myristoyl regions of proteins. Two of the peptides are hydrophobic, and the other two are hydrophilic in nature.

Hydrophobic peptides tend to aggregate in aqueous medium, and aggregates of peptides may not associate with lipid vesicles effectively (Saberwal and Nagaraj, 1993). Hence, it would be important to determine the aggregation status of peptides before their interaction with lipid vesicles is investigated. The emission maximum of the peptides at 335 nm in 31R and 14R indicates that the Trp in both peptides is shielded from aqueous environment. This shielding can occur as a result of peptide aggregation. The Trp in 31R is the N-terminal amino acid and would not be solvent-shielded if the peptide is entirely helical and in the monomeric form. However, in many short helical peptides the N- and C-terminal ends, especially 5-6 amino acids from either end, are flexible, as suggested by two-dimensional NOE experiments (Tappin et al., 1988; Zagorski et al., 1991). Moreover, in 31R the amino acids at the N terminus are not those which initiate helical structures like in model peptides (Scholtz and Baldwin, 1992). Hence, the probable conformation of 31R is a helical stretch in the hydrophobic region with a flexible N terminus. In such a conformation, the N-terminal region can loop back such that the Trp residue is proximal to the hydrophobic core. Peptide 14R has high helical content, and the molecules can aggregate in such a way that the Trp would be solvent-shielded. In 31Pal, the Trp is exposed to aqueous environment unlike in 31R. Palmitoylation thus appears to prevent aggregation. Since the fatty acid is at the N-terminal end in the peptide 14Myr, its effect is less pronounced on the aggregation properties of the peptide. The hydrophilic peptides do not aggregate, and acylation does not induce aggregation. Palmitoylation is a posttranslational event (Schmidt, 1989). In the case of proteins with hydrophobic transmembrane stretches, palmitoylation could conceivably have a role in preventing aggregation as suggested by the observation on 31Pal.

The emission characteristics of Trp residue indicate that the hydrophilic peptides 12R, 11R, and their acylated derivatives indicate weak affinity for lipid vesicles. The emission maximum at 350 nm indicates that the Trp in these peptides and their acyl derivatives are not located in the hydrophobic core of the lipid vesicles but near the polar head group region. The quenching of fluorescence in lipid environment as compared to aqueous medium could arise due to the differences in polarity on the surface of the bilayer as compared to bulk aqueous phase (Surewicz and Epand, 1984; De Kroon et al., 1990). However, the Trp fluorophore would be accessible to quenchers like I as in bulk solvent, which is reflected in the Stern-Volmer quenching constants. It is unlikely that binding of 11R, 12R and their acylated derivatives are not registered by Trp by virtue of its location near a charged amino acid. In signal sequences (McKnight et al., 1991), melittin (Dempsey, 1990), and magainin analogs (Matsuzaki et al., 1994), binding to lipid vesicles has resulted in changes in Trp fluorescence even though the position of Trp is adjacent to a charged amino acid. In the case of hydrophilic peptides, it is clear that acylation does not increase affinity to membranes or has influence in orienting the peptide chain in the lipid bilayer. In the case of hydrophobic peptides 31R and 14R, the membrane association is apparent with the acylated peptides as indicated by binding isotherms as well as quenching experiments with KI and acrylamide. The polarization values indicate that 31R and 14R also associate with lipid vesicles. The emission maximum at 335 nm suggests that the Trp is located away from the head group of the membrane bilayer.

The absence of large changes in fluorescence indicates that the environment around Trp in peptide aggregates and in lipid vesicles are similar. The Stern-Volmer plots suggest that in lipid vesicles, Trp in 14R is less accessible to aqueous quenchers than in 14Myr, indicating a more buried Trp residue. The greater polarization value of Trp in 31Pal as compared to 31R in lipid vesicles suggests a different orientation for the two peptides in lipid vesicles. In fact, the Stern-Volmer plots for 31Pal and 31R in lipid vesicles are also different, suggesting a different environment. The leveling of quenching clearly discernible in 31Pal in presence of lipid vesicles is absent in 31R.

Although our results indicate the association of 14R and 14Myr with lipid vesicles, it may not have a direct bearing on the orientation of peptide segments in membranes as myristoylated proteins do not have transmembrane spanning segments. As myristoylation does not favor binding of hydrophilic peptide segments to membranes, the assumption that myristoylation is necessary for anchoring to membranes may not be a valid one. Our results are in agreement with a recent report that the Gibbs free energy for binding of a myristoylated peptide to a phospholipid vesicle is 8 Kcal/mol, equivalent to a K of 10M, which is not sufficient for stable association of myristoylated peptide to the lipid bilayer (Peitzsch and McLaughlin, 1993; Resh, 1994). The observation that in viral proteins the fatty acid is bound to proteins (Chow et al., 1987) and the possible existence of receptors for myristoylated peptides (Resh, 1989, 1990; Goddard et al., 1989) suggest that the stable membrane-anchor function attributed to myristoylation may not be a valid one.

The polarization value, the emission maximum and quenching experiments support an orientation of 31R in the bilayer as depicted in Fig. 9A. The N-terminal end of the helix is depicted to be relatively flexible. It is unlikely that the peptide is oriented in the manner shown in Fig. 9B as quenching by I is discernible, which would not be the case were it to be oriented near the inner leaflet of the membrane. Thus, the peptide 31R corresponding to the membrane-spanning region of vesicular stomatitis virus G protein appears to insert into the lipid bilayer such that the C-terminal portion is localized near the head group region of the inner leaflet. However, for the acylated peptide we propose an orientation where the helix axis is at an angle and not perpendicular to the bilayer surface (Fig. 9C). The Trp residue would be associated with the hydrophobic portion of the bilayer and with a relatively restricted motion. Another possible orientation is shown in Fig. 9D. This structure would need reversal of peptide chain in the bilayer, i.e. formation of a helical hairpin. This model is quite unlikely as none of the amino acids in the hydrophobic core would favor a bend or chain reversal although there are two glycines present. Hence, in vivo it is likely that the modification of palmitoylation especially in proteins having transmembrane segments may serve to tilt the membrane-spanning segments from its orientation perpendicular to the bilayer surface. In fact, an orientation almost parallel to the bilayer surface is conceivable. This would also enable interaction with segments of proximal proteins in the membrane.


Figure 9: Possible modes of association of peptides 31R and 31Pal in membranes. A and B, 31R; C and D, 31Pal. The fatty acid is attached at the C-terminal portion of the peptide. In D, the two helices would be composed of approximately 13 residues unlike in A-C, where the helix corresponds to the entire hydrophobic stretch.



Based on our investigations, we would like to propose the following. (i) The primary role of myristoylation may not be an anchor for stable membrane attachment as assumed. (ii) Palmitoylation in the case of transmembrane proteins could serve to realign the transmembrane segment from an orientation perpendicular to the bilayer surface. (iii) In the case of proteins where there is no hydrophobic segment, palmitoylation may not serve as a membrane anchor and could be involved in interaction with membrane-bound proteins.

  
Table: Primary structures of peptides corresponding to fatty acylation sites in proteins and their fatty acyl derivatives

Peptide 31R corresponds to the transmembrane segment of the VSV G protein (Rose and Gallione, 1981). Peptide 12R corresponds to the C-terminal end of Ras protein (Hancock et al., 1989), 11R corresponds to the N-terminal portion of the oncogene product of HIV F/3` open reading frame, phosphorylated GTP-binding protein (Guy et al., 1987). 14R corresponds to N-terminal region of the mouse mammary tumor virus (Moore et al., 1986). Acm, acetamido methyl; Pal, palmitoyl; Myr, myristoyl.


  
Table: The emission maximum () and relative fluorescence intensity (RFI) of the peptides 31R, 31Pal, 14R, and 14Myr in urea, guanidine hydrochloride, and MeOH when excited at 280 nm

Values represent average of three independent observations. Variation was <5%.


  
Table: Fluorescence polarization values of peptides in buffer and lipid vesicles



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 91-40-672241; Fax: 91-40-671195; E-mail: nraj@ccmb.uunet.in; Telex: 0425-7046 CCMB IN.

The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; PC, phosphatidylcholine; CF, Carboxyfluorescein.


ACKNOWLEDGEMENTS

We thank C. Arunan for help in synthesis of one of the peptides. We also thank V. M. Dhople for amino acid analysis and M. V. Jaganadham for sequence analysis of the synthetic peptides.


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