helix promotion in polypeptides by polyols

Helix Promotion in Polypeptides by Polyols

JAKE BELL0

Department of Chemistry, Roswell Park Division of the Graduate School, State University of New York at Buffalo, Buffalo, New York 14263

SYNOPSIS

The helix content of [ L-LYS ( Me3) ] ,, - c104 and [ L-Lys ( Me3) 60, ~-Ala"] ,, - c l o 4 in water is markedly increased by the presence of sucrose and glycerol. For [ L-LYS ( Me3) ] ,, - C104 the ellipticity at 222 nm changes from +2 X lo3 deg cm2 dmole-' in water to -44 X lo3 in 50% glycerol. Sucrose does not promote helix formation in melittin at pH 7.2, but glycerol does. At pH 5.5 sucrose and, more so, glycerol, induce helix in melittin. Glycerol induces some helix in methylated melittin, but less than in melittin. The results are discussed in relation to excluded volume effects, AG of transfer of peptide and hydrophobic groups from water to mixed solvents, electrostatic effects, and preferential hydration. 0 1993 John Wiley & Sons, Inc.

INTRODUCTION

Sucrose and glycerol in water usually stabilize protein structure. Such stabilization has been discussed from several points of view, only a few of which will be briefly mentioned here. In these mixed solvents proteins have been reported to be preferentially hydrated.',* AG for transfer of peptide groups from water to glycerol is positive, which would tend to stabilize H-bonded structure^.^ Another mechanism of stabilization might be that of volume excl~sion.~-~ The effects of cosolvents in vitro may be relevant to the stability of proteins in vivo and of helix-forming power of the amino acid residues in vivo. The helix-forming ability of each of the amino acid residues has been studied by a variety of methods on solutions of peptides, polypeptides, or proteins in dilute aqueous solution (with dilute buffer and, usually, salt). In the cell there is a high concentration of biopolymers, which must affect the activity coef- ficients of all other components. If the transfor- mation of A + B results in a net decrease in volume, the excluded volume effect will promote the reaction as written, and vice If the volume change accompanying the incorporation of different protein

Biopolymers, Vol. 33, 491-495 (1993) 0 1993 John Wiley & Sons, Inc. CCC oooS-3525/93/030491-05

side chains into helices varies, the helix-forming po- tentials of a series of side chains may differ depend- ing on whether the transition is carried out in simple aqueous solution or in the presence of a volume ex- cluder. Or from a hydration point of view, if the differences in hydration between random coil and helix are different for the various residues, the presence of high concentrations of other molecules could alter the relative helical properties of the amino acids. The same considerations apply to other types of ordered or irregular structures.

We have made a study of the coil + helix transition in poly (L-lysine ) trimethylated on its amino group, [ L - L ~ S ( M ~ ~ ) * H C I O ~ ] , ; in a random 1 : 1 copolymer of Lys(Me3) and alanine, { [L- Lys( Me3)50, ~ - A l a ~ ' ] * HClO,},; and in melittin and methylated melittin. As cosolvents we have used glycerol and sucrose. We chose to use the synthetic polypeptides in the methylated form in order to eliminate possible changes in pK, and to be able to observe the helix + coil transition at a conveniently low concentration. [ Lys ( Me3) 1, * HClO, becomes helical (a t 0" ) at a residue concentration of 2 mM, ' whereas [ Lys], requires much higher concentra- t i ~ n . ~ - ' Melittin was selected as a natural peptide with a positive AV of helix formation, lo and methylated melittin was used because its charge is pH independent.

49 1

492 BELL0

EXPERIMENTAL

(Lys),.HBr (52.5 kDa), (Lys5', Ala5').HBr (40 kDa) , and melittin were purchased from Sigma, and NaC104 * H2O from GFS Chemicals. (Molecular weights are those supplied by Sigma.) The methylated polymers were prepared with dimethyl sulfate, as described earlier." Melittin was purified by gel filtration through Sephadex G-50, eluted with 0.01 M ammonium acetate, and freeze-dried. Melittin was methylated by the same procedure as above, and the reaction mixture was desalted on Sephadex G-15 with 0.1M ammonium acetate and freeze dried. Amino acid analysis of melittin was in conformity with its composition. Amino acid analysis of methylated melittin showed the loss of 2.95 lysine residues (theory 3) and 0.9 glycine residue (theory 1 ) . A peak for trimethyl (L-lysine) was observed (but not quantitated), and no peaks for mono- or dimethyl- lysine were seen. Glycerol was purified as described earlier." CD spectra were measured with a Jasco 500A, fitted with a water-jacketted cell holder. Temperature in the cell holder was measured with a platinum-resistance thermometer ( Omega Model 199). The concentrations of polypeptides were es- timated from absorbance at 191 nm,13 and of melittin and methylated melittin from absorbtion of the single tryptophan residue, with 6 = 5600M-' cm-' .

The solubility of benzene at 27°C was measured spectrophotometrically. A series of different excesses of benzene (Reagent grade, thiophene free) was used, and no evidence of impurities was found. Cal- ibration curves of benzene in the solvents of interest were used. The distribution of indole between water or glycerol and cyclohexane was measured at 27°C. The solutions were equilibrated in N2-flushed tubes with teflon-lined screw caps in the dark. The overall concentration of indole was varied from 1 X to 16 X 10-4M. Concentrations at equilibrium were measured spectrophotometrically, and calibration curves were made using the appropriate solvent equilibrated with the other solvent. Indole (Eastman Kodak ) was recrystallized twice from ethanol-water.

RESULTS

The effects of glycerol and sucrose on the ellipticities of [ Lys ( Me3) 1, - HC104 and [ Lys ( Me3) 5 0 ,

Ala5'], * HC104 are shown in Figure 1. [ Lys ( Me3) I n at 0.94 m M (residue) in water has a [6]222 of +2 X lo3 deg cm2 bole- ' , i.e., essentially nonhelical, while in 50% glycerol the ellipticity is -44 X lo3, that of completely helical polymer. In 20% (w/w)

Cosolvent, %

Figure 1. Ellipticity of polypeptides in aqueous sucrose and glycerol at 0-1°C. ( 1 ) [Lys(Me3)],, in glycerol; ( 2 ) [Lys(Me3)], in sucrose; ( 3 ) [Lys(Me3)", Alam], in glycerol; (4 ) [Lys(Me3)", Ala5'], in sucrose. Solvent composition: w/w for sucrose; v/v for glycerol (but the glycerol was dispensed by weight). Peptide (residue) concentration: [Lys(Me,)],, 0.94 mM; [(Lys(Me3), Ala)],, 1.1 mM. No salts or buffer used.

sucrose [ 0lZz2 = -36 X lo3 deg cm2 dmole-l. A similar experiment was done with [ Lys( Me3) 50, Alam],. At a peptide (residue) concentration of 1.1 m M in water = -12 X lo3, and in 20% sucrose -22 X lo3, 83% greater. With [ L ~ S ( M ~ ~ ) ~ ' , Ala5'], in aqueous glycerol, ellipticity rises more slowly than for [ Lys ( Me3) I n , but approaches a final value similar to that for [ Lys ( Me3) 1,. Elevated pressure shifts the random coil of (Lys), - HBr to the a-helix, l4 i.e., AV for coil + helix is negative. An excluded volume effect would be expected to operate in the same direction. Although no pressure data are available for the [ Lys ( Me3) 1, used in this work, it is not unrea- sonable that a similar effect would exist for formation of the helical core, whatever effect might be operative with regard to the side chains and the Cloy counterion.

Similar experiments were carried out with melittin, a 26-residue cationic peptide from bee venom. Melittin has no negative charges, and has a large proportion of apolar residues. Its amino acid se- quence is G-J-G-A-V-L-K-V-L-T-T-G-L-P-A-L-I- S-W-I-K-R-K-R-Q-Q-NH2. At low concentration and pH 7.2 it is a monomeric, largely random coil;

HELIX PROMOTION 493

0 0 I0 20 30 40 50 60 70 80 90 1 0 0 I10 120

[MLTI, pM

Figure 2. Ellipticity of melittin at 27°C. 0: in 36% (v / v ) glycerol; 0 in H,O, A: in 36% (w/v) sucrose. Buffer: 0.02 M sodium phosphate, pH 7.2 (measured in water).

at high pH or high ionic strength it goes over to a tetramer, with a high content of a-helix, a process promoted by increasing the peptide c~ncentration.'~ In 0.02 M sodium phosphate, pH 7.2, melittin a t 120 pM concentration has a [ 6]222 of -11 X lo3 deg cm2 dmole -', indicating partial conversion to the helix, compared with [ 0]222 of -4 X lo3 at 8 pM (Figure 2 ) . In the presence of 36% (w/v) sucrose there is essentially no change in the ellipticity of 8 or 120 pM melittin. In the presence of 36% glycerol (v/v) the ellipticity of 120 pM melittin nearly doubles to -20.6 X lo3 deg cm2 dmole-'. A t lower melittin concentrations, [ 0]222 of melittin in 36% glycerol decreases in magnitude, approaching the value of low-

concentration melittin in water. Yunes l6 reported the partial conversion of melittin to the a-helix in increasing concentrations of glycerol (up to 100% ) at a single melittin concentration (14 pM, unbuf- fered). At 100% glycerol, Yunes found a [ 01222 of

Among the possible effects of glycerol and sucrose might be changes in the pK, values of buffers and peptides. Melittin, as measured by its coil + helix conversion, exhibits a pK of about 7.5.15 Because the above experiments were done with a pH 7.2 phosphate buffer, it is possible that the greater hel- icity of melittin in glycerol might arise from a shift in pH. Therefore, we measured the CD spectrum of 40 pM melittin in a pH 5.5 phosphate buffer, well below the pK of melittin. The results (Table I ) show that, compared with the value in water, sucrose doubles the magnitude of the ellipticity, and glycerol quadruples it. But a t pH 5.5 the ellipticity (-13.3 X lo3) is somewhat less than at pH 7.2 ( -17 X lo3) .

Also, we measured CD spectra of methylated melittin, i.e., melittin with its amino groups con- verted to quaternary trimethylammonium groups, so that their positive charges are unaffected by pH. As expected, the ellipticity of methylated melittin is independent of pH from pH 2.3 to l l . 1 7 Methyl- ated melittin is much more resistant to helix formation than is melittin, and shows no concentration dependence from 30 to 290 pM in water (0.02M phosphate, pH 7.2) .17 Glycerol and sucrose increased the magnitude of the ellipticity by small amounts (Table I).

We have found that glycerol increases the solubility of benzene, by factors of 1.1 and 1.25 at 20 and 50% (v/v) , but 20% sucrose decreases the solubility to 0.89 of its value in water. From distribution experiments, AG (unitary) for transfer of indole from water to 50 and 100% glycerol was -0.96 and -2.84 kcal mole-', respectively.

-17 x lo3.

Table I in Glycerol and Sucrose

Ellipticities of Melittin and of Methylated Melittin,

[elzz2 Peptide PH" Solvent kdeg cm2 dmole-'

Melittin, 40 p M 5.5 H,O -3.3 Melittin, 40 p M 5.5 36% (v/v) Glycerol -13.3 Melittin, 40 pM 5.5 36% (w/v) Sucrose -6.7 Meth-Melittin, 120 pM 7.2 H20 -3.3 Meth-Melittin, 120 pM 7.2 36% (v/v) Glycerol -5.6 Meth-Melittin, 120 pM 7.2 36% (w/v) Sucrose -4.1

a Phosphate buffer, 0.02M.

494 BELL0

DISCUSSION

The results for [ Lys ( Me3) 1, are consistent with several mechanisms of action of sucrose and glycerol: ( 1) excluded volume {on the assumption that A V of helix formation of [ Lys ( Me3) 1, is negative, as it is for (Lys),); ( 2 ) the positive AG of transfer of peptide groups from water to mixed solvent3; ( 3 ) preferential hydration, on the assumption that these polymers are preferentially hydrated as many proteins are; and ( 4 ) promotion of polycation-counterion interaction. The lesser effect of sucrose compared with glycerol could arise in part from the fact that different concentration scales apply in Figure 1, the sucrose being w/v and glycerol v/v; thus the volume fraction of sucrose is smaller (20% v/v for glycerol is 24% w/v). Also, Winzor and Wills5 have presented work indicating that molecular size is not the only criterion for the magnitude of an excluded volume effect. The copolymer is less affected by glycerol and sucrose. If alanine is involved in hydrophobic interactions, these would contribute a positive AV18 counteracting the - A V of the lysine residues. This is consistent with an excluded volume effect. The thermodynamics of transfer of methyl groups from water to aqueous glycerol would be im- portant, but data are not available for this. A few data are available for other hydrophobes of interest in respect to proteins. Transfer of the tyrosine side chain from water to 50% glycerol is negative, -493 cal mole-', but for the valine side chain is near zero, +11 cal mole-' (and -74 cal mole-' in 99% glycerol) .3 For the peptide group (plus one CH2 group), based on glycylglycine less glycine, AG of transfer from water to 50% glycerol is +158 cal mole-' (+367 for 99% glycerol) ? AG for peptide groups would tend to stabilize hydrogen-bonded structures, while AG for apolar groups would have unpredictable effects. Glycerol increases the solubility of benzene and indole, sucrose decreases the solubility of benzene. Lakshmi and Nandi reported a positive AG for transfer of hydrophobes from water to 2 M sucro~e.'~ Because both cosolvents affect the ellipticity of the synthetic polypeptides in the same direction but have opposite effects on the solubility of some hydrophobes, it may be that hydrophobic effects are not the cause of increased helix formation; this, however, requires further study. Timasheff et al., in considering the stabilization of tubulin in aqueous sucrose and glycerol, noted that these polyols have opposite effects on the surface tension (increased by sucrose and decreased by glycerol) ." Therefore,

surface tension cannot account for the results with methylated lysine and lysine-alanine copolymer.

The results for melittin are also difficult to explain because of the complexity of the process, involving formation of the H-bonded a-helix and the hydrophobic interactions between the four chains of melittin that assemble into the tetramer." The promotion of helix by glycerol (and by sucrose at pH 5.5) is inconsistent with an excluded volume effect. The work of Thompson and Lakowicz shows a + A V for formation of the helical tetramer." The net A V includes a solvent contribution; in the case of hydrophobic interaction ( i.e., removal of hydrophobes from water), A V is positive." However, in the presence of a substantial amount of a cosolvent, the solvent contribution to A V could change. Also, A V for the hydrophobic effect with liquid hydrocarbons (the usual experimental subjects) would be more positive than for tight hydrophobic packing common for protein packing. Promotion of helix is consistent with the positive AG for transfer of peptide groups from water to glyceroL3 As noted above for lysine polymers, we do not know how the thermodynamics of transfer of the several types of apolar side chains would affect the association of monomers to tetramer that is involved in stabilization of the helix.

Melittin takes up a monomeric a-helical state in solvents like methanol and e than01. '~ ,~~-~~ But that is not what is happening here. As the melittin concentration is reduced (at constant glycerol concentration), [ 8]222 decreases in magnitude, indicating that dissociation to less ordered monomer takes place on dilution, as it does more gradually in water. Thus, a t 120 pM in 36% glycerol melittin is helical and is most likely a tetramer. Yunes suggested that melittin associates in glycerol.16 Because Yunes worked at a constant low melittin concentration ( 14 p M ) and at glycerol concentrations up to loo%, it is possible that helix formation in that case may have taken place in the monomer. Thus, at low melittin concentration a monomeric helix may exist at high glycerol concentration, and at high melittin concentration a tetrameric helix may exist at 36% glycerol. The formation of tetrameric melittin in 36% glycerol indicates that AG for transfer of the apolar side chains, all but one of which are aliphatic, to this solvent is not negative overall (although it probably is negative for tryptophan).

The reason for the near-absence of effect of sucrose on melittin is also unclear. The positive A V of formation of tetrameric helical melittin leads one to expect a decrease in tetrameric helix by the excluded volume effect. The positive AG for transfer

HELIX PROMOTION 495

of hydrophobic groups from water to aqueous sucrose would be expected to promote tetrameric helix formation. Perhaps both effects are present and coun- teract each other. Another possible effect on helix promotion is electrostatic. The positive charges in- hibit formation of the tetrameric helix, and can be overcome by high salt, with specific ion effectsz3 in- dicative of ion binding, high pH, or acylation of the amino groups.15 Sucrose or glycerol will affect both inter- and intrachain repulsions and counterion binding. A factor that increases repulsions is likely to promote counterion binding; these would probably have opposite effects on formation of the tetrameric helix. Similar considerations would apply to the lysine polymers.

The lower magnitude of [ 8]22z for melittin in water in pH 5.5 buffer than in pH 7.2 buffer is expected, because a t pH 7.2 the coil + helix transition is un- derway.15 At pH 5.5 sucrose and glycerol have greater helix-inducing power than a t pH 7.2. The very small effect of 36% glycerol on methylated melittin compared with native melittin may have several causes. First, a larger positive AV for methylated melittin than for melittin might result from the methyl groups; this would oppose an excluded volume contribution. Second, ion binding may differ. At any event the greater resistance (from whatever cause) of methylated melittin in water to form the tetrameric helix persists in the mixed solvents.

The results presented here show that effects of cosolvents on helix-forming tendency are complex and may involve a number of phenomena, including preferential hydration, excluded volume, electrostatic effects and thermodynamic effects on polar and apolar groups, surface energy, and solvent structure (which affects all the other properties). These are not all likely to act in the same direction, and will vary from cosolvent to cosolvent. Within a cell, although polyols are present only in low concentration, the high concentrations of other sub- stances can exert the various effects discussed above. These factors might, in principle, change the relative helix-forming propensities of the amino acids as compared to in vitro gradations. Further, in these experiments we have addressed only peptides that form a-helices. But glycerol and sucrose stabilize natural proteins that also contain other types of structures, both regular (such as /3-structures) and nonregular.

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Received May 27, 1992 Accepted July 24, 1992

helix promotion in polypeptides by polyols

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