From the
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
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
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(
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 Me
On-line formulae not verified for accuracy
The emission spectra of the
peptides 12R and 31R and their palmitoylated derivatives are shown in Fig. 1. The emission maximum is
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
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 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.
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
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
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
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
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.
Values represent average of three independent observations.
Variation was <5%.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(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.
Materials
)
-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.
SO,
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
NaS
O
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).
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.
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.
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.
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.
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.
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.
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.
of 10
M, 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.
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
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
Table: Fluorescence polarization values of
peptides in buffer and lipid vesicles
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.