©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Amphipathicity in the Folding, Self-association and Biological Activity of Multiple Subunit Small Proteins (*)

(Received for publication, July 29, 1994; and in revised form, October 11, 1994)

Enrique Pérez-Payá Richard A. Houghten Sylvie E. Blondelle (§)

From the Torrey Pines Institute for Molecular Studies, San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect that altering amphipathicity has on the folding process and self association of melittin, a small model protein, has been investigated using single amino acid substitutions of lysine 7, a residue distant from the contact residues involved in the hydrophobic core of tetrameric melittin. While substitutions of such a residue were not expected to interfere with the packing process, the largest alterations in the potential overall amphipathicity of melittin were found to prevent the folding into an alpha-helical conformation to occur and, in turn, to prevent the self association. Amphipathic alpha-helices were found to be a key determining feature in the early folding process of the self association of peptides and protein segments. Those substitutions, which prevented the inducible amphipathic folding ability, were also found to result in a loss in hemolytic and antimicrobial activity. These results, combined with studies of the binding to artificial liposomes and to polysialic acids, indicate that the losses in activity were due to an initial inability to be induced into an amphipathic alpha-helix and to self associate. Ultimately, melittin's self association is proposed to be required to penetrate the carbohydrate barrier present in biological membranes.


INTRODUCTION

Amphipathic alpha-helices are a ubiquitous structural feature found in many proteins and biologically active peptides. This structural motif has been found to play multiple roles in protein folding, protein-protein recognition, protein-membrane interactions, and protein and peptide biological activity. Amphipathic alpha-helices, in turn, have a pronounced tendency to self associate in defined structural arrangements (for review, see (1) ). However, due to the complexity and compound variables involved in such interactions, the identities of the amino acid residues and/or sequences that contribute to the folding, self association, stability, and activity of the native proteins are not well understood. In an effort to study this problem, a number of groups using protein mutants (2) and guest peptides(3, 4, 5) are investigating the propensity of individual amino acid residues to induce specific secondary structures, regardless of the resulting alteration of the amphipathicity of peptide and protein domains.

The role and importance that altering the amphipathicity plays in relation to the folding process was studied in the present work using melittin, a model, small tetrameric self assembling protein. Melittin is a 26-residue peptide (GIGAVLKVLTTGLPALISWIKRKRQQ-NH(2)), which is known to adopt an amphipathic alpha-helical conformation in the presence of biological membranes, micelles, and surfactants(6) . Furthermore, at high peptide concentration or at high ionic strength, melittin is known to self associate into a tetrameric structure driven by the formation of a hydrophobic core(7) . Reduction of electrostatic repulsion between the positive charges occurring at high pH and/or high salt concentration was also found to favor the self association of melittin(7, 8) . Variations of residues distant from the hydrophobic core and clearly solvent-exposed are not expected to interfere with the protein packing process and, in turn, should result in the formation of a similar self aggregational state. Thus, the lack of self association following substitution or omission of specific residues in a peptide sequence would tend to imply that the alteration of the amphipathicity of the modified region and/or the nature of the mutation are important elements in the early stages of the folding process. Using x-ray crystallography, the lysine at position 7 in melittin's sequence has been shown to be fully solvent exposed following tetrameric self association(9, 10) , and its microenvironment has been shown to be essentially unaltered upon tetramerization(11) . Single substitution analogues at position 7 with amino acids having varying chemical properties were synthesized in the present study to determine the effect of such alterations on melittin's folding and self association capability, using circular dichroism (CD) spectroscopy.

In addition to its importance in the folding process, the role of amphipathic alpha-helices self association on melittin's and other peptides' biological activity remains unclear. Thus, the relationship between the tetrameric self assembly of melittin or mastoparan in solution and their lytic activities has not been defined, nor has the role of the trimerization of glucagon. The results obtained in the current structural studies using melittin's substitution analogues were therefore related to the differences in the biological activities (hemolytic and antimicrobial activity) of these substitution analogues in order to elucidate the effect amphipathic alpha-helices have on the self association in relation to the biological activity of peptides.


EXPERIMENTAL PROCEDURES

Peptide Synthesis

Peptides were prepared by simultaneous multiple peptide synthesis using t-butoxycarbonyl chemistry as described elsewhere(12) . Final cleavage and deprotection were carried out using a ``low-high'' hydrogen fluoride procedure using a 24-vessel cleavage apparatus ((13) , Chiron Mimotopes Peptide Systems, San Diego, CA). The peptides were then purified by preparative reverse phase HPLC (^1)using a DeltaPrep 3000 reverse phase HPLC combined with a Foxy fraction collector (Millipore Corp., Waters Division, San Francisco, CA). Analytical reverse phase HPLC and laser desorption time-of-flight mass spectroscopy (Kratos Kompact MALDITOF mass spectrometer, Kratos, Ramsey, NJ) were used to determined the purity and identity of the peptides.

Size Exclusion Chromatography

Size exclusion chromatography of the purified synthetic peptides was performed on a Ultraspherogel SEC 2000 (Beckman, Fullerton, CA) size-exclusion column run isocratically in low salt (0.05% trifluoroacetic acid, 5 mM MOPS) or high-salt (1.5 M NaCl, 5 mM MOPS-NaOH, pH 7.0) buffer at a flow rate of 0.1 ml/min. Prior to injection, samples were filtered using 1-µm Acrodisc CR PTFE filters (Gelman Sciences, MI). Samples were applied to the column at 0.5-1 mg/ml. Bovine serum albumin, carbonic anhydrase, cytochrome c, and insulin chain B (Sigma) were used as standards.

Preparation of Phospholipid Liposomes

A mixture of egg phosphatidylcholine (egg PC) and phosphatidylserine (PS, Sigma) (10 mg) was dissolved in a chloroform/methanol (9:1 (v/v)) mixture and dried by a stream of nitrogen gas. The dried lipid was hydrated in 2 ml of 5 mM MOPS-NaOH buffer, pH 7.0, with repeated vortex mixing for 15 min. The suspension was sonicated in an ice-water bath for 20 min using an ultrasonic generator equipped with a microtip probe (Vibra cell, Sonics and Materials, Inc., Danbury, CT).

Circular Dichroism Measurements

All measurements were carried out on a Jasco J-720 circular dichroism spectropolarimeter (CD, Eaton, MD) in conjunction with a Neslab RTE 110 waterbath and temperature controller (Dublin, CA). The CD instrument was routinely calibrated with an aqueous solution of D-10-camphorsulfonic acid at 290.5 nm. CD spectra were the average of a series of three to seven scans made at 0.2-nm intervals. CD spectra of the same buffer and/or lipid solutions without peptides were used as baseline in all of the experiments. Ellipticity is reported as mean molar residue ellipticity []; the limits of error of measurements at 222 nm were ± 500 (deg cm^2 dmol). The secondary structure of the peptides was estimated using the curve fitting procedure described by Yang et al.(14) . Although this algorithm is based on the CD spectra of known secondary structures of proteins, this method can be used for the comparison of the secondary structures of sequence-related peptides. For salt-induced aggregation, liposome binding studies, and sialic and colominic acid titrations, stock solutions were separately prepared with: 1) peptide in buffer (5 mM MOPS-NaOH, pH 7.0); 2) 5 M NaCl; 3) 5.6 mM phospholipid egg PC/PS (8% PS in mol) or egg PC/PS/cholesterol (33% cholesterol, 5% PS in mol); 4) sialic acid (1 mg/ml); and 5) colominic acid (1 mg/ml). The samples studied were then prepared by mixing the appropriate solutions at a range of defined ratios. Samples were tested for nonspecific peptide aggregation or peptide-induced liposome aggregation using a Hewlett Packard 8452A diode array UV spectrophotometer (Palo Alto, CA). Peptide concentrations were determined by UV spectrophotometry at 280 nm in buffer and in the presence of 7.2 M guanidine HCl; the reported extinction coefficient standard for melittin in buffer ( = 5570 M cm; (15) ) was used as reference.

The concentration dependence of the CD spectra of the peptides in 5 mM MOPS-NaOH buffer at pH 7.0 was analyzed for size-defined oligomer formation, nM &cjs0635; L, K = [M]^n/[L], where [M] and [L] are the monomeric and self-assembled concentrations of the peptides, respectively, n is the degree of association, and K is a dissociation constant. The experimentally determined mean molar ellipticity/residue at 222 nm for each peptide at varying concentrations was analyzed using the formalism proposed by Taylor and Kaiser (16) with the software Graphpad (ISI, San Diego, CA).

The guanidinium hydrochloride denaturation studies were carried out by preparing mixtures of a stock solution of peptide in buffer (5 mM MOPS-NaOH, pH 7.0), buffer alone, plus a solution of 7.2 M guanidine HCl in buffer. The ratios of buffer and 7.2 M guanidine HCl solutions were varied to give the appropriate final guanidine HCl concentrations. The samples were allowed to equilibrate for 30 min at room temperature prior to CD measurement. The thermal dependence of ellipticity at 222 nm was analyzed using the program TTSCAN (Jasco) with a step resolution of 0.1 °C and a temperature slope of 20 °C/h. The ellipticity values obtained from both, guanidine HCl and thermal denaturation studies were analyzed using the software Graphpad.

Hemolytic and Antimicrobial Assays

The hemolytic activities of the peptides were determined using human red blood cells (RBCs). The blood was collected in heparin, maintained at 4 °C, and used either the same day or the following day. The cells were washed three times with phosphate-buffered saline (35 mM phosphate buffer, 0.15 M NaCl, pH 7.0) and resuspended in phosphate-buffered saline. Neuramidase (Sigma) was added to half of the samples for a final concentration of 10 IU/ml(17) . Both the neuramidase-treated and untreated control samples were incubated at 37 °C for 2 h. The neuramidase and liberated sialic acids were washed away with isotonic sucrose, and the resulting pellets were retained. The hemolytic activity of the peptides were determined as described (18) using 96-well tissue culture plates. In brief, 100 µl of a 0.5% RBC solution were added to an equal volume of each peptide in phosphate-buffered saline. The plates were incubated for 1 h at 37 °C, and the optical density of the supernatant was measured at 414 nm. The concentration of peptide necessary to lyse 50% RBCs (HD) was then determined for each peptide using a sigmoidal curve fitting method (Graphpad).

Microdilution assays were carried out against Escherichia coli ATCC 25922 and E. coli BAS 849 in 96-well tissue culture plates as described elsewhere(19) . Following overnight incubation at 37 °C, the optical density at 620 nm of the bacterial suspension in Muller-Hinton broth was measured in the presence of serial 2-fold dilution of peptides. The concentration of peptide necessary to inhibit 50% of the bacterial growth (IC) was then determined for each peptide using a sigmoidal curve fitting method (Graphpad).


RESULTS

alpha-Helical Propensity of Melittin's Substitution Analogues

Melittin and eight substitution analogues at position 7 were prepared using simultaneous multiple peptide synthesis ((12) ; sequences listed in Table 1). As determined by CD spectroscopy, random structure with a characteristic minimum near 200 nm was found in aqueous buffer for melittin and all of the substitution analogues except subK7E, which adopted a 40% alpha-helical conformation (Table 1). In order to confirm the ability of these peptides to adopt an alpha-helical conformation, the CD spectra of each peptide of this series were measured in the presence of trifluoroethanol, a solvent known to induce helicity in single-stranded potentially alpha-helical polypeptides(20) . In 60% trifluoroethanol, the ellipticities at 222 nm of all of the analogues (including the parent sequence) were found to have values of approximately -25,000 ± 2,000 deg cm^2 dmol, except for subK7A and subK7E, which had slightly higher values (Table 1). The ellipticity values determined at 222 nm were found to be independent of the peptide concentration (up to 600 µM, data not shown), which suggests that the helical structure in each peptide is unimolecular in this environment as reported for other amphipathic peptides(21) .



Aggregation Studies

Peptides that have the potential to form amphipathic alpha-helical structures in many instances aggregate in a highly cooperative manner in aqueous solution. In such an aggregate, the hydrophobic domains of amphipathic helices generally interact to form a hydrophobic core. In order to evaluate the concentration dependence of this self association process of melittin and its analogues, their CD spectra were analyzed at varying peptide concentrations in 5 mM MOPS, pH 7.0, at 25 °C. At low peptide concentration, only subK7E showed a partial alpha-helical conformation ([] = -13,400 deg cm^2 dmol; Fig. 1A). This may be due to tight intermolecular interactions stabilized by electrostatic effects in a manner similar to reported coil coiled structures(3) . At elevated peptide concentrations (concentration greater than 0.15 mM), the ellipticity values found at 222 nm for those peptides with a positive or negative charge at position 7 are indicative of an alpha-helical protein (Fig. 1A). Those analogues lacking a charge at position 7 failed to aggregate in the range of concentrations tested (Fig. 1B). This lack of self aggregation correlates with a decreased hydrophobic moment relative to the parent sequence (Table 1). The significant self association of melittin, subK7D, and subK7E can be best explained by the formation of a tetramer. The dissociation constants found for these peptides correspond to free energies of tetramerization of -17.0, -17.9, and -18.3 kcal/mol, respectively. The free energy of tetramerization obtained for melittin is similar to the previously reported value of -17.7 kcal/mol obtained in phosphate buffer(22) , and higher than the value obtained in Tris buffer (-15.4 kcal/mol; (8) ). This suggests that, as described in the case of phosphate buffer(22) , MOPS buffer stabilizes the tetrameric form of melittin.


Figure 1: Concentration-dependent ellipticities at 222 nm. The CD spectra were recorded in MOPS buffer at varying peptide concentrations (from 4 to 500 µM). [] is plotted for A, , melittin; , subK7D; and &cjs2132;, subK7E; B, +, subK7A; times, subK7G, circle, subK7I; , subK7L; box, subK7V; and up triangle, subK7W. Computer generated monomer-tetramer curves are shown superposed on the data.



It has been shown that melittin is in an unfolded state at micromolar concentrations under conditions of low ionic strength, but adopts a tetrameric helical structure under conditions of high ionic strength (7, 23) . Folding of the eight melittin substitution analogues as a function of ionic strength was therefore investigated further (Fig. 2). In agreement with the concentration-dependent results described above (Fig. 1A), melittin, subK7D, and subK7E were found to fold into an alpha-helical conformation with increasing NaCl concentration (Fig. 2A). The NaCl concentration required to induce 50% folding ([NaCl]) was 0.73, 0.47, and 0.15 M for melittin, subK7D, and subK7E, respectively. The CD spectra of each peptide in the presence of varying NaCl concentration exhibited an isodichroic point at 203 nm, indicating a two-state equilibrium between a random coil and an alpha-helix. Furthermore, the shape of each analogues' CD spectra in the presence of a high salt concentration indicates the presence of alpha-helical content.


Figure 2: Ionic strength-dependent ellipticities. The CD spectra were recorded in MOPS buffer for a peptide concentration of 20 µM in the presence of NaCl. [] is plotted for A, , melittin; , subK7D; and , subK7E; B, +, subK7A; times, subK7G; circle, subK7I; , subK7L; box, subK7V; and up triangle, subK7W.



In contrast to the concentration dependent behavior of the three analogues described above (Fig. 1B), two distinctly different behaviors were observed for the remaining six analogues (Fig. 2B). It should be noted that these six peptides differ from the preceding ones by their lack of a charged amino acid at position 7. The alpha-helical content of subK7A and subK7G increased with increasing ionic strength (Fig. 2B), but not with increasing peptide concentration ([NaCl] = 0.5 and 1.0 M, respectively). On the other hand, in the presence of 1 M NaCl, the CD spectra of subK7I, subK7L, subK7V, and subK7W indicated a predominantly beta-sheet structure (minimum around 217 nm; the shape of the CD spectra is similar to the recently reported spectra for a predominantly beta-sheet protein(24) ). Minimal or a lack of alpha-helicity was indicated by the absence of a clearly defined minimum at 208 nm.

The degree of self assembly of the peptides was also investigated using size exclusion chromatography. Two separate solvent systems were used to mimic the environment used in our CD studies: 5 mM MOPS containing 0.05% trifluoroacetic acid in order to minimize the nonspecific interactions between the peptides and the chromatographic support (no change in conformation for melittin appeared in CD upon decreasing the pH); and 5 mM MOPS-NaOH buffer, pH 7.0, containing 1.5 M NaCl. Both conditions are expected to favor self association. The elution times of the nine peptides, as well as a range of molecular weight standards, are shown in Table 2. In 5 mM MOPS, 0.05% trifluoroacetic acid, all of the peptides had elution times similar to insulin chain B, indicating that they were monomeric. The presence of a minor peak (for subK7I, subK7L, and subK7V) was found at an elution time close to the void volume of the column and is believed to indicate the presence of large nonspecific aggregated structures. In the presence of 1.5 M NaCl, however, melittin, subK7A, subK7D, subK7E, and subK7G appear to be tetramers, as described for melittin(9, 10) . Only monomeric forms were indicated in both chromatographic systems for the other four peptides (subK7I, subK7L, subK7V, and subK7W).



Denaturation Studies Using Guanidinium Hydrochloride

The denaturation curves were determined at 25 °C by monitoring the peptides ellipticities at 222 nm as a function of the concentration of guanidine HCl. These studies were initially carried out for melittin, subK7A, subK7D, subK7E, and subK7G, peptides found earlier to self associate at high peptide concentration or high ionic strength ( Fig. 1and Fig. 2). The minimum concentration in NaCl that ensured the presence of the tetrameric structures was used to compare the stability of tetrameric forms of these analogues (1.2 M NaCl for subK7A, subK7D, and subK7E, and 2 M NaCl for melittin and subK7G; Fig. 3A). The peptide concentrations for the five peptides were equal (20 µM) to rule out the possible concentration effects on stability. Significant differences in the guanidine HCl concentration transition midpoint values ([guanidine HCl], concentration at which the helical content is 50% of the maximum helical content in the absence of denaturant) were found among these peptides (Table 3). The highest [guanidine HCl] was found for subK7E (2.13 M), which is in sharp contrast to the value of 0.54 M found for subK7G. These variations in the indication of aggregation stability are noteworthy when one takes into account the fact that these analogues differ by only 1 amino acid.


Figure 3: Guanidine HCl denaturation curves. The CD spectra were recorded for a peptide concentration of 20 µM in 5 mM MOPS buffer. A, containing 1.2 M NaCl for +, subK7A; , subK7D; and , subK7E; and 2 M NaCl for , melittin; and times, subK7G; B, containing 1.2 M NaCl (without NaCl in the inset for subK7I (circle), subK7L (), subK7V (box), and subK7W (up triangle). Computer generated sigmoidal curves are shown superimposed on the data.





In a monomer-to-tetramer transition, the unfolding process should follow a two-state equilibrium model between folded tetramers (F(4)) and unfolded monomers (U): F(4) &cjs0635; 4U, with K = [U]^4/[F(4)] = 4Pt3(fu4/(1 - f(u))(3, 7) . K represents the tetramer dissociation constant; P(t) represents the total peptide concentration; and f(u) represents the molar fraction of unfolded peptide as determined from the ellipticity at 222 nm. The formula f(u) = ((n) - )/((n) - (u)) is used to calculate f(u), where represents the observed ellipticity at a given guanidine HCl concentration, and (n) and (u) are the ellipticities of the tetrameric folded and monomeric unfolded states, respectively. The value K was calculated in the transition zone for each guanidine HCl concentration tested. The K were found to increase logarithmically with guanidine HCl concentration for the five peptides (mel, subK7A, subK7D, subK7E, and subK7G). These results suggest the presence of an invariant tetramer structure, which, upon increasing guanidine HCl concentration, vary in stability.

The free energy variation in the unfolding process (DeltaG(u)) was determined at each guanidine HCl concentration using the formula DeltaG(u) = -RT lnK. The free energy of the unfolding process in the absence of denaturant (DeltaGuH(2)O) was estimated by linear extrapolation of the DeltaG(u) values to a guanidine HCl concentration equal to zero using the equation DeltaG(u) = DeltaGuH(2)O - m [guanidine HCl](25) . The DeltaGuH(2)O and m values for all the peptides are listed in Table 3. Based on the crystal structure of tetrameric melittin(9, 10) , position 7 is fully exposed to the solvent. If one envisions that the stability of a four-bundle or tetrameric melittin array results solely from the hydrophobic interactions occurring in the hydrophobic core, one would expect to find the same stability for all the peptides studied. However, at 25 °C in the absence of denaturant, tetrameric self assembly of subK7E was found to be approximately 3 kcal/mol more favorable than the tetrameric self assembly of melittin. SubK7E also had the highest transition midpoint, indicating greater stability. In contrast, subK7G had the lowest transition midpoint ([guanidine HCl] = 0.54 M), which can be related to its elevated m value. As proposed by Matouschek et al.(25) , an m value of this magnitude suggests a significant exposure to the solvent of subK7G on denaturation.

The denaturation curves for subK7I, subK7L, subK7V, and subK7W were determined in 5 mM MOPS-NaOH buffer, pH 7, in the presence or absence of 1.0 M NaCl. As shown in Fig. 3B, the denaturation process was virtually independent of the initial buffer conditions. In all cases, the [guanidine HCl] values were found to be approximately 3 M (Table 3). These results, combined with those derived from the CD aggregation studies and size exclusion experiments described above, require a different analysis of the guanidine HCl denaturation curves for these peptides. If one assumes that the intrachain interactions or the orientation of given amino acid residues are determinant factors in the folding of these analogues, then the unfolding reaction should follow a two-state equilibrium model between folded and unfolded monomers: F &cjs0635; U, with K(u) = [U]/[F] = f(u)/(1 - f(u))(26) . K(u) represents the equilibrium constant for the unfolding process, and f(u) represents the molar fraction of unfolded peptide as determined from the ellipticity at 222 nm described above (similar K(u) values were obtained using f(u) values generated from ellipticities at 217 nm). The free energy of unfolding in the absence of denaturant (DeltaGuH(2)O) was estimated in a similar manner by linear extrapolation of DeltaG(u) at each guanidine HCl concentration to a final guanidine HCl concentration equal to zero (Table 3). The four analogues were found to have virtually identical stability.

Thermal Stability

The thermal stability of melittin and its substitution analogues was investigated by monitoring the changes in ellipticity at 222 nm as a function of temperature. In agreement with the above mentioned studies, the thermal behavior shows that the nine peptides can be separated into three different groups. The first group included those peptides that have a charged amino acid at position 7. The second group was made up of subK7A and subK7G; both showed salt-induced alpha-helical conformation but remained monomeric and unstructured at a high peptide concentration in NaCl-free buffer. Finally, the third group was made up of those peptides having a bulky hydrophobic amino acid at position 7, which did not fold either in high ionic strength or at high peptide concentration. Fig. 4illustrates representative examples of the effect of temperature on the ellipticity at 222 nm for one peptide from each of these groups.


Figure 4: Thermal stability. The ellipticity at 222 nm of each analogue was recorded at a peptide concentration of 20 µM in 5 mM MOPS buffer containing 1.2 M or 2 M NaCl as described in Fig. 3, upon first increasing the temperature from 4 to 90 °C (solid line) and then, using the same sample, decreasing the temperature from 90 to 4 °C (dashed line) for A, subK7D; B, subK7G; and C, subK7W.



For melittin, subK7D, and subK7E (first group of peptides), the CD spectral changes seen upon thermal denaturation in the presence of NaCl reflect the occurrence of a transition from a salt-induced folded state to an unfolded state. This denaturation process appears to be almost completely reversible upon lowering the temperature back to its initial value (Fig. 4A). No significant cold denaturation was observed upon cooling from 25 to 4 °C for melittin and its analogues under our experimental conditions (2 M NaCl for melittin and subK7G, and 1.2 M NaCl for the other analogues). The smooth cooperative transition obtained for these three peptides under the conditions studied suggests the occurrence of a thermodynamic equilibrium(27, 28) . The calculated T(m) values for helix-coil transition (i.e. the temperature at which 50% of the peptide is in its unfolded form) was approximately 60 °C, indicating a stable alpha-helical array for these peptides (Table 3).

Cooperative denaturation profiles were also observed for subK7A and subK7G (Fig. 4B) when these peptides were heated from 4 to 90 °C. The lower T(m) values found for subK7A and subK7G relative to the first group of peptides (41 and 43 °C respectively) reflect a pronounced loss of thermal stability. Furthermore, the initial folded state was not recovered upon lowering the temperature from 90 to 4 °C; rather a nonspecific aggregation process was observed.

Finally, for those peptides that have a bulky hydrophobic amino acid at position 7, the change in ellipticity as a function of temperature was almost linear, indicating a noncooperative denaturation process (Fig. 4C). These results can be understood by the peptides' lack of folding into alpha-helices in aqueous solution(29) .

Binding Studies to Phospholipid Liposomes

While the peptides had different structures in aqueous solution, studies on their binding to lipids were necessary to confirm that the induced secondary structures observed in aqueous solution were related to their biological activity. As in the thermal studies, the CD spectra of the nine peptides can be classified in the same three groups described above. Thus, upon increasing the ratio [phospholipid]/[peptide] (R) using egg PC/PS (92:8) small unilamellar vesicles (SUVs), a gradual increase in alpha-helical content was observed for subK7D, subK7E, and melittin (as illustrated in Fig. 5A for subK7E). In contrast, subK7A and subK7G showed a low alpha-helical content for the range of lipid concentrations tested (as illustrated in Fig. 5B for subK7G). Finally, at low R, the shape of the CD spectra of those analogues having a bulky hydrophobic group at position 7 revealed the presence of varying degrees of beta-structure (as illustrated in Fig. 5C for subK7I). At R values higher than 30, these peptides predominantly showed alpha-helical structures.


Figure 5: CD spectra in the presence of vesicles for (A) subK7E, (B) subK7G, and (C) subK7I. The CD spectra were recorded at a peptide concentration of 20 µM in 5 mM MOPS buffer in the presence of egg PC/PS with R equal to 0 (solidline), 4 (dottedline), 20 (dashedline), and 50 (dot-dashline).



The binding of each analog to SUVs was quantitatively analyzed using spectral enhancement(30) . The ellipticity enhancement is defined as ( - (o))/(o), where is the ellipticity at 222 nm for the peptide in the presence of SUVs and (o) is the ellipticity for R = 0. The ellipticity of the fully bound peptide ((max)) and the lipid concentration at which half of the peptide is bound to the vesicles (L) were determined using the equation: ( - (o))/(o) = [((max) - (o))/(o)] [L]/([L] + L), where [L] is the concentration of lipid (Table 4). Thus, subK7E was found to have a high binding affinity to SUVs, which resulted in 76% alpha-helical content at R = 16 (value corresponding to 50% melittin bound to the SUV) as estimated using the curve-fitting procedure describe by Yang et al. ((14) ; Table 4). At this R value, 45 and 37% alpha-helical content was estimated for melittin and subK7D, respectively. All of the other analogues showed approximately 20% beta-structure under these conditions. At R = 50, none of the peptides showed significant beta-structure content (Table 4).



The initial driving force for melittin's interaction with phospholipid membranes is generally thought to be an electrostatic interaction between the highly positively charged C-terminal of melittin and the negatively charged phospholipid head groups(31) . Since each of the peptides studied here have the same unmodified positively charged C-terminal, they should all show similar affinity to phospholipid membranes if the primary binding force is due to electrostatic interactions. However, each of the peptides has different L values, as well as different alpha-helical content upon binding to SUVs (Table 4). Thus, it is unclear if these differences are due to the method used to determine the binding affinity, since it is assumed for the calculation of the L that the peptides fold into an alpha-helical conformation upon binding to SUVs. As an alternative approach, multilamellar vesicles (not sonicated) were used to determine the binding affinity of melittin and three analogues (subK7D, subK7G, and subK7L). Although this method is not as accurate as for the determination of affinity through the use of SUVs, and even though multilamellar vesicles have slightly different physical properties than SUVs, this method allows for the evaluation of the percent of each peptide that did not bind to the vesicles regardless of the peptide folding propensity. Thus, at an R of 16, the percentage of nonbound peptide ranged from 9 to 30% (Table 4), which suggests that the amount of peptide bound to the vesicles is virtually the same for melittin as for the three analogues. These results can be interpreted if one assumes that, for a determined peptide density on the bilayer surface, a complex equilibrium between peptide-lipid, peptide-peptide, and peptide-aqueous solvent exists, and furthermore that the peptides' ability to disrupt the phospholipid bilayer depends on which of these interactions is the predominant one. The finding of a high alpha-helical content at R = 50, i.e. when the peptide surface density was lowered, which, in turn, means that peptide-lipid interactions were dominant, supports these premises.

Finally, the ability of melittin and its analogues to bind to cholesterol present in biological membranes was investigated using egg PC/PS/cholesterol (62:5:33, mol/mol/mol) liposomes. Overall, lowered affinity parameters (i.e. higher L values; Table 4) were found in the presence of cholesterol relative to the L values obtained using SUVs that did not contain cholesterol. Only subK7W had a similar L in the presence of SUVs with and without cholesterol, which may be due to the known ability of tryptophan residues to assume a defined orientation when binding to cholesterol (32) .

Conformational Changes of Melittin and Its Analogues in the Presence of Colominic Acid

The conformational changes of melittin induced by high pH(33) , high salt(7) , and high peptide concentration (34) , as well as by the addition of surfactants (35) and/or lipid(36) , have been extensively studied. Recently, Takeda and Moriyama (37) reported conformational changes in melittin upon interaction with polyglutamic acid polymers. In a related manner, we examined the conformational changes of melittin and its analogues induced by naturally occurring polymers of N-acetylneuraminic acid (referred to as polysialic acids), which are present on the surface of biological cells. The negative charges of the polysialic acid groups reportedly make no contribution to the electric potential at the external bilayer-aqueous interface on erythrocyte membranes, since the charged groups are at a significant distance from this interface and are fully exposed to the aqueous phase(17) . This charge array can therefore be considered as a first barrier preventing the interaction of polypeptides with cell membranes.

The CD spectra of melittin and its analogues were measured in the presence of colominic acid (poly-2,8-N-acetylneuraminic acid) at different concentration ratios (R = C/P; where C represents the colominic acid concentration, and P represents the peptide concentration in mg/ml). Melittin was induced into an alpha-helical conformation upon increasing R to 1, with no further changes in ellipticity found upon increasing R beyond 1 (Fig. 6A). A small amount of turbidity appeared at ratios lower than 1. In contrast, no helix formation was observed for melittin in the presence of the monomeric sialic acid (data not shown). Interestingly, the presence of a negative charge at position 7 in subK7E or subK7D did not affect the induced conformation in the presence of negatively charged colominic acid relative to melittin (i.e. increases in helicity upon increasing R; Fig. 6B). SubK7E showed higher alpha-helical content than melittin and subK7D under these conditions in a manner similar to that found in the presence of salt or SUVs. An alpha-helical conformation was also observed for subK7G and subk7W in the presence of colominic acid. In contrast, the shape of the CD spectra of subK7A, subK7I, subK7L, and subK7V revealed a significant beta-structure content, with, however, a much wider band at the beta-structure minima (Fig. 6C), which suggests that other structures such as a distorted alpha-helix were also present.


Figure 6: CD spectra in the presence of colominic acid for (A) melittin, (B) subK7E, and (C) subK7I. The CD spectra were recorded at a peptide concentration of 20 µM, in 5 mM MOPS buffer, in the presence of colominic acid at ratio R = 0 (solidline), 1 (dottedline), 2 (dashedline), and 6 (dot-dashline).



Biological Activity

Melittin is well known for its lytic activity on cell membranes, especially those of RBCs(38) . Kinetic studies have shown that monomeric melittin binds to RBCs within seconds and induces the release of hemoglobin(39) . The ``hemolytic dose'' of the nine peptides necessary to lyse 50% of the cells (HD) indicated that only subK7I, subK7L, and subK7V have little or no hemolytic activity (Table 5). This result is in agreement with earlier studies of the importance of melittin-inducible amphipathicity on its lytic activity(18, 23, 40, 41, 42, 43) . These poor hemolytic activities can be related to the low folding propensity found for the peptides by CD under all of the conditions described above. Furthermore, the three peptides were found to adopt partial beta-structures, which would prevent the occurrence of membrane perturbation as proposed for omission analogues of melittin having low hemolytic activity(23) . However, the binding affinities to SUVs of these three peptides (L; Table 4) are higher than the binding affinity of subK7G and similar to that of subK7A, even though both of these analogues have significant hemolytic activity. In order to determine if the charged sugars (i.e. polysialic acids) on the erythrocyte surface were responsible for the differences in hemolytic activity of melittin and its substitution analogues, the hemolytic activity of each analog was determined using RBCs lacking polysialic acid, i.e. treated with neuramidase. As shown in Table 5, the three analogues, subK7I, subK7L, and subK7V became highly active toward neuramidase treated RBCs, while no change in activity was observed for the other peptides. These results suggest that the poor hemolytic activity found for these analogues was at least in part due to their inability to reach the cell membranes.



The antimicrobial activity against E. coli was also determined for melittin and its analogues using two different strains, a strain used in standard assays (ATCC 25922) and a drug more permeable mutant strain (BAS 849). In a manner similar to the activity against treated RBCs, the three peptides (subK7I, subK7L, and subK7V) exhibited no activity against the standard strain of E. coli, while they became highly active against the mutant strain (Table 5). It is also noteworthy that, while subK7W was as active as melittin against the untreated RBCs, it had lower antimicrobial activity against the standard strain of E. coli. This may be due to the occurrence of tryptophan/cholesterol interactions with RBCs, as proposed above in the study of the binding affinity of this analog to SUVs containing cholesterol. The absence of cholesterol in bacterial membranes may account for these differences in hemolytic and antimicrobial activities.


DISCUSSION

The effects of altering the degree of amphipathicity on the folding process, as well as the membrane binding ability of peptides, was examined in the present work using closely related model peptides. Since position 7 of melittin's sequence is fully exposed to the solvent in melittin tetramer and, in turn, is distant from the hydrophobic core(9, 10) , the use of single substitution analogues at position 7 was expected to provide insight into the early steps of the folding and self association processes. Thus, differences in the overall stability of the self-assembled tetramers resulting from such substitutions should be attributable to changes in the initial coil to helix transition and not in the interhelical packing interactions. Furthermore, this alteration affects only the first alpha-helix in folded melittin, while the hinge, second alpha-helix, and the basic C-terminal remain unchanged for all the analogues. This was confirmed by the adoption of a fully alpha-helical conformation by all of the analogues in the presence of trifluoroethanol.

Two approaches are commonly used to study folding in amphipathic peptides: variation in peptide concentration and induction by increasing salt concentration. An increase in peptide concentration results in the folding of amphipathic peptides, driven by hydrophobic interactions, which in turn are favored by the closer proximity of peptide molecules. On the other hand, folding in the presence of increased salt concentration is predominantly driven by the weakening of charge repulsions. In agreement with the hydrophobic moment values (<µ(H)>), only those analogues that remained amphipathic when induced into an alpha-helix were found to fold when their concentration was increased (i.e. subK7E and subK7D). The free energies of tetramerization in salt-free buffer for subK7D and subK7E (-17.9 and -18.3 Kcal/mol, respectively; -17.0 kcal/mol for melittin) appear to be consistent with an electrostatic stabilization in an anti-parallel arrangement between the negative charge at position 7 and the basic C-terminal region. Preliminary molecular modeling studies, based on the crystal structure of tetrameric melittin (9) and generated from the crystallographic dimer (Protein Data Bank entry 2MLT) using symmetry and residue replacement operations, suggested that electrostatic interactions may occur between glutamic acid-7 and the positively charged residues of the C terminus of the anti-parallel chain of the same dimer. In contrast, in the case of subK7D, such interactions appeared to be more favorable between aspartic acid-7 and the basic C-terminal residues of the equivalent chain in the second dimer. These potential differences in the amino acids involved in such interactions may be responsible for the higher stability observed for the tetrameric form of subK7E. As determined by the guanidine HCl denaturation studies, the presence of NaCl was found to stabilize the tetrameric structure of these two analogues, as well as that of melittin, by more than 7 kcal/mol. These results are in agreement with the earlier reported stabilization of tetrameric melittin by 5-6 kcal/mol in the presence of 0.5 M NaCl(8) . These results suggest that besides the hydrophobic interhelix stabilizing interactions, the amino acids located in the N- and C-terminal regions in anti-parallel peptide bundles are involved in overall protein stability.

While the two analogues subK7A and subK7G remained random at high peptide concentrations, they folded upon increasing the NaCl concentration. Both analogues were also found to have lower hydrophobic moments than melittin, subK7E, or subK7D, and higher changes in entropy than melittin as determined by the van't Hoff method (DeltaDeltaS = 62 cal/Kbulletmol and 152 cal/Kbulletmol for subK7A and subK7G relative to melittin, respectively). It appears that these losses in amphipathicity are compensated by the decrease in charge repulsion that occurred at high ionic strength. This results in an increased propensity for folding induction and is supported by the occurrence of an additional folding energy in the presence of salt similar to the one calculated for subK7E and subK7D. The poor folding capability of subK7A and subK7G was confirmed by their nonreversible thermal denaturation. Upon lowering the temperature, these peptides aggregated in a disordered manner at temperatures higher than the T(m) values, as evidenced by precipitation. However, the relationship between their inability to refold upon decreasing the temperature and their inability to fold upon increasing peptide concentration is uncertain.

In contrast to the above peptides, no tetrameric folding was observed for subK7I, subK7L, and subK7V at either high peptide concentration or high ionic strength. These results indicate that the intermolecular hydrophobic interactions are not strong enough to compensate for the energetically unfavorable positioning of these hydrophobic amino acids in the hydrophilic face of the alpha-helix. A small amount of distorted beta-structure was instead observed in the presence of salt. We believe this results mainly from the occurrence of intramolecular hydrogen bonding, as determined by size exclusion chromatography. These results show that amphipathicity is a determining feature in the early folding process, i.e. in the induction of the appropriate molten globule or intermediate structures, and, in turn, in the self association ability of peptides or protein segments.

Artificial liposomes are commonly used as a model system to study the binding affinity of peptides or proteins to cell membranes(44, 45) . However, the binding affinity to SUVs found for melittin's substitution analogues could not be directly related to the variation in their hemolytic or antimicrobial activities. The peptide density on the aqueous/lipid interface also appears to be involved in the adoption of the active structure, as indicated by the increase in folding capability observed upon increasing the SUV/peptide ratio (R). The differences observed in folding ability in the presence of salt and in the presence of SUVs for those analogues having hydrophobic residues at position 7 lead to the conclusion that an ionic strength-dependent feature was responsible for the lack of activity found for these peptides. The increases in activity observed for these peptides upon removing the charged sugars from the RBCs, combined with their lack of folding in the presence of colominic acid, support this hypothesis. The sugar present in biological membranes can thus be seen as assisting in the induction of self association (tetramerization in the present case), which would prevent hydrophobic residues from being exposed to the sugar charges. Such structures can be expected to be stabilized by the surrounding high ionic strength. The aggregate would then pass through this first biological barrier to reach the phospholipid bilayers, and, finally, may act on the membrane following a still controverted mechanism (either in an aggregate form or in a folded monomeric form(6, 46, 47) ).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 45583 and by a NATO Postdoctoral Fellowship (to E. P. P.). 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.: 619-455-3803; Fax: 619-455-3804.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; egg PC, egg phosphatidylcholine; PS, phosphatidylserine; MOPS, 4-morpholinepropanesulfonic acid; RBC, red blood cell; SUV, small unilamellar vesicle.


ACKNOWLEDGEMENTS

We thank Ema Takahashi and Edward Brehm for technical assistance, and Eileen Silva for editing this manuscript.


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