(Received for publication, May 22, 1995; and in revised form, June 9, 1995)
From the
Fatty acid acylation is a posttranslational modification found
in membrane proteins that have hydrophobic sequences serving as
transmembrane segments as well as those that do not have them. The
fatty acids myristate and palmitate are linked through an amide bond to
N-terminal glycine and SH of cysteine via a thioester bond,
respectively. In order to elucidate whether or how fatty acid acylation
would modulate peptide structure, especially in hydrophobic
environment, we have carried out circular dichroism studies on
synthetic peptides both hydrophobic and hydrophilic in nature,
corresponding to fatty acylation sites and their fatty acyl
derivatives. The hydrophilic peptides were 12 residues in length
as studies on proteins modified by site-directed mutagenesis indicated
that a peptide segment of
12 residues is sufficient to direct
acylation as well as membrane association, especially when the fatty
acid is myristic acid. The peptide corresponding to a transmembrane
segment composed of 31 residues as well as its palmitoyl derivative was
found to adopt
-helical structure. Acylation appeared to favor
increased partitioning into micelles even in the case of a hydrophobic
peptide. The hydrophilic peptides and their myristoyl or palmitoyl
derivatives showed very little ordered structure in micelles. Our
results suggest that the myristoyl and the palmitoyl moieties do not
have the ability to ``force'' a hydrophilic peptide segment
into a hydrophobic micellar environment. Thus, the mere presence of a
fatty acid moiety may not be sufficient for membrane binding and
recycling as is assumed especially in proteins in which no hydrophobic
segment is present.
It is now becoming increasingly clear that small, linear peptide
fragments of proteins, in aqueous solution, can exhibit defined
conformational preferences for structures other than random-coil
structure(1, 2, 3, 4, 5, 6, 7, 8) .
Many structures like nascent helix(1, 2) , reverse
turns(4, 5) , and ordered helix (6) have been
characterized in short peptides. In fact, a 9-residue peptide
corresponding to the internalization signal of low density lipoprotein
receptor was found to adopt a reverse turn conformation in solution,
correlating well with the proposal that internalization signals in
coated pit receptors is a -turn conformational
motif(4, 5) . This suggests that inference of
structure drawn from studies on isolated short peptides could have
relevance in biological processes. Apart from examining conformational
preferences in water, the solvent trifluoroethanol (TFE) (
)has been used extensively in studying the conformation of
peptides, especially while examining helix propensities(9) .
The solvent stabilizes the helical structure in peptides. In circular
dichroism studies, this solvent is particularly useful in the case of
peptides that exhibit limited solubility in water.
Fatty acid
acylation is a posttranslational modification found in membrane
proteins which have hydrophobic sequences serving as transmembrane
segments as well as in proteins like Ras p21, which is relatively
hydrophilic with almost all of its structure in the
cytoplasm(10, 11, 12) . The fatty acids,
myristate and palmitate, are linked through an amide bond to N-terminal
glycine and SH of cysteine via a thioester bond, respectively. In order
to elucidate whether or how this fatty acid acylation would modulate
peptide structure especially in hydrophobic environment, we have
carried out circular dichroism studies on synthetic peptides
corresponding to fatty acylation sites in proteins and their fatty acyl
derivatives (Table 1). We have synthesized peptides with myristic
acid at the N-terminal glycine and with palmitic acid at the cysteine
residue. The peptide sequences were chosen from fatty acylated proteins
having hydrophobic membrane-spanning segments as well as hydrophilic
sequences. Peptide 31R corresponds to the transmembrane segment of the
vesicular stomatitis virus G protein(13) , whereas peptides
12R, 14R, and 11R correspond to acylation sites in oncogene proteins.
Peptide 12R corrresponds to the C-terminal end of the Ras
protein(14) , 14R corresponds to N-terminal region of the mouse
mammary tumor virus(15) , and 11R corresponds to the N-terminal
portion of the oncogene product of HIV F/3` orf, phosphorylated
GTP-binding protein(16) . Studies on proteins modified by
site-directed mutagenesis (17, 18) as well as chimeric
proteins (19) have indicated that peptide segments of 12
residues are required to direct acylation as well as membrane
association, especially when the fatty acid is myristic acid. Hence we
have chosen peptides composed of 11-14 residues, except in the
case of 31Pal where the transmembrane spanning region has been
included, resulting in a peptide composed of 31 residues.
The peptides with the exception of 31R and 31Pal were purified by fast performance liquid chromatography on a reverse phase pep RPC 5/5 (Pharmacia) column. Peptides 31R and 31Pal were purified by thorough washing with methanol and acetonitrile. Both the peptides exhibited limited solubility in these solvents and could be easily separated from the impurities which were soluble in methanol and acetonitrile The purity and composition of the peptides were determined by amino acid analysis on a LKB 4151 Alpha Plus amino acid analyzer, after hydrolysis in vacuo with trifluoroacetic acid, 6 N HCl (1:2). The purity of the nonacylated peptides was further confirmed by sequencing on 473A protein sequencer (Applied Biosystems). The presence of fatty acids was confirmed by analysis on a Hewlett-Packard 5840A gas-liquid chromatograph, after hydrolysis.
All peptide stock solutions were
made in methanol (MeOH) and dimethyl sulfoxide (MeSO) and
were quantitated by OD at 280 nm and quantitative amino acid analysis.
Aliquots of required concentrations of peptide stock solution were
transferred to test tubes and dried in vacuum. Spectroscopic grade TFE
was added to make up the required concentration just before recording
the spectra. For experiments in micelles, appropriate volumes and
concentrations (above the critical micellar concentration of 8 mM sodium dodecyl sulfate (SDS) and 0.64% octyl glucoside (OG)) of
detergent solution were added after drying the sample in a test tube,
prior to recording of the spectra. The concentrations of SDS and OG for
the experiments were 20 mM and 1% (w/v), respectively.
Spectra were deconvoluted by convex constraint analysis as described by Perczel et al.(26) . For all the spectra, 15 iterations were done.
The conformations of peptides were examined in TFE, a solvent known to promote helical conformation in peptides with helical propensity(2, 6) , micelles of SDS which provide a simple hyrophilic/hydrophobic interface mimicking a membrane environment, octyl glucoside, a non-ionic detergent, and dioleoly phosphatidylcholine lipid vesicles.
Figure 1: CD spectra of peptides 31R and 31Pal. a, 31R in TFE; b, 31R in SDS micelles; c, 31Pal in TFE; d, 31Pal in SDS micelles. Peptide = 0.05 mM.
Figure 2: Pure components obtained after convex constraint analysis of the CD spectra of peptides 31R and 31Pal. a, 31R in TFE: curve I, 74%; curve II, 32%. b, 31R in SDS: curve I, 66%; curve II, 33%. c, 31Pal in TFE: curve I, 66%; curve II, 34%. d, 31Pal in SDS; curve I, 85%; curve II, 15%.
The
spectra of 31R and 31Pal in OG micelles and results of spectral
deconvolution are shown in Fig. 3. Peptide 31R shows a single
minimum 216 nm. A pure component with contribution of
72% has
very similar appearance to the experimentally obtained spectrum. The
component contributing
28% is not characteristic of helix, turn,
or
-sheet conformations. The
value of
35,000 at the minimum argues against a
-sheet conformation
that would result in
= -18,000.
However, distorted helices (27) do exhibit a single minimum
with
< -18,000. Hence it appears that the
structure of 31R is that of a distorted helix in neutral micelles. The
spectrum of 31Pal is typical of a helical structure that is also
reflected in the ``pure'' component spectra.
Figure 3: CD spectra of peptides 31R and 31Pal in octyl glucoside micelles and the pure components obtained after convex constraint analysis of the CD spectra. A, 31R: curve I, 28%; curve II, experimental spectra; curve III, 72%. B, 31Pal: curve I, 25%; curve II, experimental spectra; curve III, 75%. Peptide = 0.05 mM.
The spectra
of 12R and 12Pal in TFE and SDS are shown in Fig. 4. The
spectrum of 12R in TFE shows a minimum at 200 nm and a shoulder at
225 nm. The
values and the crossover that would
be below 195 nm argue against the peptide existing in a predominantly
ordered conformation. However, the spectra do not resemble those of
``classical'' random structure(28) . The peptide has
the sequence TPGC, which could form a
-turn(29, 30) . In SDS, although the general
appearance is similar to that in TFE, the
value at
the minimum is considerably more. The spectra of the acylated peptide
12Pal in TFE and SDS micelles are shown in Fig. 4, c and d. In TFE, the spectrum shows a minimum at
215
nm with a crossover at
197 nm. The spectra may be assigned to a
-turn conformation with residues TPGC participating in the turn.
Acylation, here, seems to increase the proportion of peptide in a
-turn conformation. The spectrum in SDS is very much similar to
that in TFE. Results of the convex analysis of CD curves shown in Fig. 4are presented in Fig. 5. The spectra of 12R in TFE
yield four pure components (Fig. 5a). Curve I is characteristic of unordered conformation and is present to an
extent of
41%. Curves II and III may be
assigned to
-turn conformations. Their contributions to the
structure are 33 and 11%, respectively. Curve IV cannot be
unequivocally assigned to
-helix,
-sheet, or
-turn
conformation. In SDS, Fig. 5b indicates a random
component of
44%. Although curves II and III cannot be unambiguously assigned, component IV may be assigned to
-turn conformation. Fig. 5c indicates a decrease
in the extent of random conformation for 12Pal in TFE. The pure spectra
indicate a
-turn contribution of 52% (i.e. curves II and III). Thus, palmitoylation at the middle of the
peptide chain seems to favor
-turn formation. Surprisingly,
acylation does not appear to favor
-turn conformation in the
micellar environment of SDS as the spectrum (Fig. 4d)
is very similar to 12R in SDS (Fig. 4b). The spectra of
12R and 12Pal in OG micelles were characteristic of peptides in
unordered conformation. This suggests that although acylation would
result in increased hydrophobicity of the peptide and consequently more
favorable partitioning into micelles, it does not happen. Hence, the
mere presence of the fatty acid may not be sufficient for association
with membranes especially when the peptide chain in the vicinity of the
acylation site is hydrophilic.
Figure 4: CD spectra of peptides 12R and 12Pal. a, 12R in TFE; b, 12R in SDS micelles; c, 12Pal in TFE; d, 12Pal in SDS micelles. Peptide = 0.1 mM.
Figure 5: Pure components obtained after convex constraint analysis of the CD spectra of peptides 12R and 12Pal. a, 12R in TFE: curve I, 41%; curve II, 33%; curve III, 11%; curve IV, 10%. b, 12R in SDS: curve I, 44%; curves II+III, 39%; curve IV, 18%. c, 12Pal in TFE; curve I, 36%; curve II, 20%; curve III, 16%; curve IV, 21%.
Figure 6: CD spectra of peptides 14R and 14Myr. a, 14R in TFE; b, 14R in SDS micelles; c, 14Myr in TFE; d, 14Myr in SDS micelles. Peptide = 0.1 mM.
Figure 7:
Pure components obtained after convex
constraint analysis of the CD spectra of peptides 14R and 14Myr. a, 14R in TFE: curve I, 46%; curve II, 28%; curve III, 5%. b, 14R in SDS: curve I, 48%; curve II, 44%. c, 14Myr in TFE; curve I,
39%; curve II, 24%; curve III, 13%; curve
IV, 15%; curve V, 9%. d, 14Myr in SDS; curve
I, 33%; curve II, 68%; curve III, 19%; curve
IV, 16%. Inset in c and d correspond to
components having extrema between -30 and
+10.
The spectra of 11R in TFE and SDS are
shown in Fig. 8, a and b. It is evident that
only a fraction of the peptide exists in ordered conformation. The
small fraction of ordered conformation could conceivably be a
-turn structure. The spectra of the myristoylated peptide 11Myr
shown in Fig. 8, c and d. are very similar to
those of the nonacylated peptide. Results of convex constraint analysis
of the CD spectra in Fig. 8are shown in Fig. 9. The pure
components and weights indicate predominantly unordered conformation
(
52%). The curves indicate that the residual ordered conformation
consists of
-turns. Myristoylation does not appear to be
sufficient for partitioning of 11Myr into micelles. CD spectra
indicated that peptides 11R and 11Myr did not partition into OG
micelles.
Figure 8: CD spectra of peptides 11R and 11Myr. a, 11R in TFE; b, 11R in SDS micelles; c, 11Myr in TFE; d, 11Myr in SDS micelles. Peptide = 0.1 mM.
Figure 9: Pure components obtained after convex constraint analysis of the CD spectra of peptides 11R and 11Myr. a, 11R in TFE; curve I, 46%; curve II, 27%; curve III, 21%. b, 11R in SDS; curve I, 45%; curve II, 18%; curve III, 19%. c, 11Myr in TFE; curve I, 25%; curve II, 45%; curve III, 19%. d, 11Myr in SDS; curve I, 51%; curve II, 23%; curve III, 17%.
Peptides 31R, 31Pal, 14R, and 14Myr yielded spectra in micelles as shown in Fig. 1and Fig. 6only when the concentration of the detergent was above the critical micellar concentration. Below critical micellar concentration precipitation was observed. Satisfactory CD spectra could not be obtained in lipid vesicles composed of dioleoyl phosphatidylcholine due to intense scattering. The CD spectra were independent of the peptide concentrations in the range of 0.025 mM to 0.2 mM, indicating the absence of aggregation in this concentration range.
The conformation of peptides corresponding to acylation sites
in proteins have been examined by circular dichroism in TFE a solvent
of low dielectric constant, stabilizing -helical structures and
micelles of SDS and OG. Micelles of SDS offer a simple hydrophilic-
hydrophobic interface and have been used extensively as a mimic of
membrane active peptides. The spectra were deconvoluted into their pure
components by the method of Perczel et al.(26) . This
has aided in revealing structural features not easily discernible by
visual inspection of the spectra. Peptide 31R adopts an
-helical
structure in a medium of low dielectric constant as would be expected
of a peptide corresponding to a transmembrane region. While 14R tends
to adopt helical conformation, 12R and 11R are largely unordered with a
fraction of peptide possibly occurring in distorted helical or
-turn conformation. The crystal structure of the Ras protein shows
an
-helix structure in the C-terminal region(31) .
Although detailed structures of the parent proteins of 11R and 14R are
not available, the homologous proteins show preference for helical or
-structure. Thus, the acylated regions do not appear to have a
common defined structural motif. Our results indicate that effects of
acylation on structure are variable. In the case of 11R, there is no
drastic structural difference from the nonacylated peptides, even in
hydrophobic environment. In 14R, myristoylation results in
conformational heterogeneity. In 31R, acylation results in a slight
increase in helical content in SDS micelles, whereas in 12R,
palmitoylation seems to stabilize a
-turn, but only in TFE. No
increased partitioning into micelles is discernible.
The crystal structures of the myristoylated catalytic subunit of cAMP-dependent protein kinase has been determined recently (32) and in this structure, the myristoyl chain is intimately associated with the polypeptide chain of the protein. The surface of the enzyme surrounding the myristoyl moieties attached to the N-terminal glycine is very hydrophobic. The recombinant enzyme obtained from Escherichia coli, lacking the myristoyl group, was more labile to heat denaturation, indicating that the myristoyl group has an important role in conferring structural stability to the protein. A structural role is also evident in the poliovirus capsid protein, VP4, where the myristoyl group provides a hydrophobic anchor between subunits on the coat surface(33) . Our results suggests that the myristoyl chain or even the palmitoyl chain does not appear to have the ability to force a hydrophilic peptide segment into a hydrophobic micellar environment. Thus, the presence of a single myristoyl chain may not be sufficient for membrane association of a globular protein. In fact, the Gibbs free energy for binding of a myristoylated protein to the lipid bilayer indicates that it is not sufficient to stably anchor a myristoylated protein to the lipid bilayer(34) . In a class of G proteins involved in vesicular trafficking, the proteins switch between a cytosolic form in which the myristoyl fatty acid chain is in association with a protein and a form where the fatty acyl chain helps in membrane association(35) . This switching phenomenon would argue against the myristoyl chain providing a stable membrane anchor. Thus, it is unlikely that the myristoyl group has a primary role in providing an anchor for membrane association.
The increased helical content when 31Pal is associated with micelles indicates that palmitoylation increases affinity of 31Pal to micelles. Thus, in proteins where palmitoylation occurs in the vicinity of a transmembrane segment, it may favor greater partitioning of hydrophobic segment into the membrane and may have a role in the assembly of membrane proteins. Very little is known as to how palmitoyl groups modulate peptide structure especially in non-membrane-associated proteins. In G protein-coupled receptors that have covalently attached palmitoyl group, signal transduction proceeds through acylation-deacylation cycles(36, 37) , indicating a dynamic role for palmitoyl modification. The palmitoyl group could also have a structural role similar to that of the myristoyl group as the palmitoyl chain too does not appear to force a hydrophilic peptide segment into a hydrophobic mileu. Thus, the mere presence of a fatty acid moiety may not be sufficient for membrane binding especially in proteins which do not have a hydrophobic peptide segment in the vicinity of the acylation site. There must clearly be additional factors that have a role in membrane association of fatty acylated proteins, as there is increasing evidence that this modification is a dynamic one with acylation-deacylation cycles, especially in proteins involved in signal transduction(37) .