Proc. Natl. Acad. Sci. USAVol. 86, pp. 5286-5290, July 1989Biochemistry

Unusually stable helix formation in short alanine-based peptides(model a-helix/protein folding/helix-coil transition)

SUSAN MARQUSEE, VIRGINIA H. ROBBINS, AND ROBERT L. BALDWINDepartment of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305.

Contributed by Robert L. Baldwin, April 17, 1989

ABSTRACT Short, 16-residue, alanine-based peptidesshow stable a-helix formation in H20. This result is surprisingwhen contrasted with the classical view that regards the a-helixas a marginally stable structure in H20 and considers shorthelices unstable. The alanine-based peptides are solubilized byinsertion of three or more residues of a single charge type,lysine (+) or glutamic acid (-). The results cannot be explainedby helix stabilization resulting from concentration-dependentassociation or by the interaction of charged residues with thehelix dipole. Our results are not predicted by the parametersfor alanine and lysine that have been determined by the"host-guest" method: these parameters predict that a 16-residue peptide should not show measurable a-helix formation.Analysis of the role of the hydrophobic interaction in a-helixformation [Richards, F. M. & Richmond, T. (1978) in Molec-ular Interactions and Activity in Proteins, Ciba FoundationSymposium 60, ed. Wolstenholme, G. E. (Excepta MedicaAmsterdam), pp. 23-25] does not show an unusually stronghydrophobic interaction in a helical block of alanine residues.The likely explanation for our results is, therefore, that indi-vidual alanine residues have a high helical potential. It is not yetknown whether any other amino acids show this property, andthe origin of this property is also unknown.

The a-helix is the most abundant element of secondarystructure in proteins (1); yet the a-helix has been regarded inthe past as only marginally stable in H20. Studies of a-helixformation by long polypeptides, together with the Zimm-Bragg model, predict (2) that short protein fragments as wellas other peptides should not show measurable helix forma-tion in H20. The Zimm-Bragg helix-coil transition theoryrelates the helix content ofa polypeptide to three parameters:s, the intrinsic helix-forming propensity of an amino acid; o,the constant for nucleating the helix; and n, the number ofpeptide units in the polypeptide. This model neglects se-quence- and position-dependent side-chain interactions.Studies with polypeptides, termed "host-guest" experi-ments, determine the Zimm-Bragg parameters for an indi-vidual amino acid by incorporating it randomly into a helix-forming polymer such as poly(hydroxybutyl-L-glutamine).Although the determined values of s for the 18 different aminoacids studied by the host-guest method (3) are significantlydifferent, these values are all close to 1 (0.6-1.3 at 20°C),values indicative of marginal helical stability even for longpolypeptides. Experimental studies give values for a _10-3(4). These parameters indicate that no peptide <20 residuesin length should exhibit measurable helix formation in H20 atany temperature if the Zimm-Bragg model applies (2).Whereas most reports do not show observable helix con-

tent in short protein fragments and other peptides, there arenow several examples in which helix formation has beendetected. The C- and S-peptide fragments of ribonuclease A(residues 1-13 and 1-20, respectively) were the first examples

of this kind (5-8). At low temperature (30C) and pH 5, the Cpeptide was found to have -25% helix content (6). Recentlywe showed that simple peptide sequences of de novo design,containing chiefly alanine with inserted pairs of glutamic andlysine residues, can form quite stable a-helices (9). Theinserted pairs ofglutamic and lysine residues are spaced threeor four amino acids apart to permit ion-pair formation, andhelix stabilization by ion pairs is clearly observed with the"i+4" spacing. In all examples to date of short peptidehelices in H20, helix stabilization by sequence- or position-specific side-chain interactions, such as ion-pair and chargedgroup-helix dipole interactions, is strongly implicated. It isnot clear, however, whether all of the unexpected helicalstability in these peptides can be attributed to specific side-chain interactions. Therefore, we ask what helix contentthese short peptide sequences would show in the absence ofside-chain interactions.

In this study, our goal is to determine the helix-formingproperties of individual residues in short peptides, beginningwith alanine. We show here that helix formation is striking inpeptides that contain only alanine plus a small number ofresidues of a single type of charged amino acid (lysine orglutamate). The resulting helices are monomolecular, and thedesign of the peptides is such that the results cannot beexplained by ion-pair formation or by charged group-helixdipole interactions. Our results indicate that alanine itselfcanstabilize short helices. This finding clashes with the classicalview that, because hydrogen bonds between the peptidegroup and H20 compete favorably with helical peptide hy-drogen bonds (10), the a-helix is intrinsically a marginallystable structure in H20. Moreover, the Zimm-Bragg param-eters for alanine obtained by the host-guest method aresmall, s = 1.07 and o- = 8 x 1O-4 (200C) (11), and suggest thatshort alanine peptides should not show observable a-helixcontent in H20 (2).

MATERIALS AND METHODSPeptide Design. Because most helix-favoring amino acids

are hydrophobic, special attention is needed in the design ofpeptides to ensure that they are H20-soluble. Alanine isfound frequently in protein helices but oligo(L-alanine) pep-tides are not H20-soluble. Ooi and coworkers (12) havestudied a single block of (Ala)20 solubilized by an adjacentblock of (Glu)20. In our designed peptides, charged residuesare inserted within a short alanine block to solubilize it. Whenthis approach is used, it is important to avoid sequences thatform amphiphilic helices and also sequences in which hy-drophobic and hydrophilic residues alternate because thesesequences tend to form P-sheets (13, 14). An amphiphilichelix tends to self-associate along the hydrophobic face ofthehelix. Thus, Ho and DeGrado (15) have designed peptidesthat form amphiphilic helices and self-associate to formfour-helix bundles. The helix is stabilized by association,

Abbreviation: TFE, trifluoroethanol.

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3K (I) Ac-A A A A K A A A A K A A A A K A - NH2

3K (11) Ac- A K A A A A K A A A A K A A A A - NH2

4K Ac- A K A A K A A A A K A A A A K A - NH2

6K (I) Ac-A KA AK AK A A KA K A A K A - NH2

6K (11) Ac- A K A A A K K A A A K K A A A K A - NH2

3E Ac- A E A A A A E A A A A E A A A A - NH2

(i+3) E,K Ac- A E A A K A E A A K A E A A K A - NH2

(i+4) E,K Ac- A E A A A K E A A A K E A AAKA -NH2

FIG. 1. Sequences of the six alanine-based peptides comparedwith sequences oftwo peptides designed to allow ion pair formation.Peptides were also synthesized with tyrosine at position 1 forconcentration determination (see Materials and Methods). Ac, ace-tyl; A, alanine; K, lysine; and E, gluamic acid.

which can be measured from the concentration dependenceof helix stability.The peptides studied here have sequences based on our

previous design of helix-forming peptides that have thepotential to form ion pairs (9). Fig. 1 illustrates the peptidesequences studied. The sequences of two of the originalion-pair peptides are also listed for comparison. To measurethe helix-forming properties of an individual amino acid, it isnecessary to avoid helix stabilization by ion pairs and otherspecific interactions between side chains. Thus, the solubi-lizing residues used here belong to a single charge type, eitherlysine or glutamic acid. The designed peptides contain 16 (or17) residues-chiefly alanine-with 3 to 6 charged residuesinserted. The charged residues spiral around the a-helix toavoid forming an amphiphilic helix and to help solubilize theotherwise insoluble block of alanine. Association or aggre-gation can be tested by studying the concentration depen-dence of the helix-coil transition, and the results are com-pared with data for four-helix bundles (15) and coiled-coildimeric helices (16).Charged groups can interact with a nearby pole of the helix

dipole; this interaction will be helix-stabilizing when the twoare of opposite sign and helix-destabilizing when they are oflike sign. This phenomenon has been termed a "chargedgroup-helix dipole" interaction. Two types of tests havebeen made here to determine whether charged group-helixdipole interactions are solely responsible for helix formationin these peptides. (i) Two different peptides have beendesigned with three solubilizing lysine residues, 3K(I) and3K(II). The peptide 3K(II) contains a lysine near the Nterminus or positive pole of the helix dipole, whereas 3K(I)

contains a lysine near the C terminus or negative pole. (ii)Charged group-helix dipole interactions are also evaluatedby examining the pH dependence of helix formation.

Peptide Synthesis. All peptides except for 3K(II) weresynthesized by solid-phase peptide synthesis on a DuPont2100 coupler with conventional N-tert-butyloxycarbonyl (t-Boc) chemistry (17). Peptide 3K(II) was synthesized on aMilligen 9050 synthesizer using 9-fluorenylmethoxycarbonyl(FMOC) chemistry. Couplings were monitored by the Kaisertest (17), repeated if necessary, and finally capped with aceticanhydride. Peptides were synthesized as C-terminal amideson p-methylbenzhydrylamine (polystyrene/1% divinylben-zene) resin. To determine peptide concentrations quantita-tively, all peptides were synthesized with a tyrosine atposition 1 and, for comparison, a second set was made withalanine at position 1. Because solid-phase peptide synthesisproceeds from the C terminus toward the N terminus, thesetwo sets of peptides are easily synthesized by simply remov-ing half the resin before the final N-terminal coupling cycle.Peptides synthesized by the t-Boc method were cleaved fromthe resin using a modified "low-high" trifluoromethanesulfo-nic acid-cleavage protocol (18).

Peptide Purification. Peptides were purified first by gelfiltration chromatography on a Sephadex G-25sfcolumn in 10mM ammonium acetate, pH 4.75, and then by reverse-phasechromatography on C18 resin by using a gradient of 0-40%oacetonitrile in 0.1% trifluoroacetic acid. Peptide purity andcomposition were determined by fast protein liquid chroma-tography (Pharmacia) and amino acid analysis. The primary-ion molecular weight was determined by fast-atom bombard-ment mass spectrometry.

Circular Dichroism (CD) Measurements. CD spectra weretaken on an Aviv 6ODS spectropolarimeter. Samples wereprepared as described (9). To determine peptide stock con-centration accurately, the set of peptides with tyrosine atposition 1 was used. Stock peptide concentration was thendetermined by measuring tyrosine absorbance in 6 M guani-dine hydrochloride at 275 nm (e75 = 1450) (19). CD mea-surements on the set of peptides containing alanine at posi-tion 1, such as pH dependence of helix formation, are thenscaled to match the signal obtained with the correspondingpeptide containing tyrosine 1. The assumption that the twopeptides will show the same CD intensity at wavelengthswhere the tyrosine side chain does not contribute to thespectrum and at pH values where the tyrosine is not ionizedis based on the finding (20) that in the C-peptide helix residue1 is frayed and makes little contribution to helix stability.

In our previous studies, peptide concentration was deter-mined by ninhydrin analysis of hydrolyzed peptide samples.Measuring the tyrosine absorbance was found to be both

7o -5E0)a) -10-a

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CY)

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I FIG. 2. (a) CD spectrum of the

peptide 3K(I) (20 ,M) in 1.0 MNaCl (pH 7.0, 1.0°C). (b) Thermalunfolding of the peptide 3K(I) asmeasured by -[10222, the mean

80 residue ellipticity at 222 nm (0.01M NaCl, pH 7.0).

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Table 1. Helix content at pH 7 and 1.0"C-(l222 (Cm2degdmol-1)

Peptide 0.01 M NaCi 1.0 M NaCl

3K(I) 22,900 25,1003K(II) 21,300 24,8004K 17,300 22,7006K(I) 6,100 13,6006K(II) 8,300 18,0003E 22,600 21,000

more accurate and more precise than the ninhydrin method.Results obtained by ninhydrin analysis were on average10-20%/o lower than those obtained by tyrosine absorbance(this corresponds approximately to 10-20% higher values ofmean residue ellipticity). Therefore, values from our previ-ous studies are probably too high and are less accurate thanthose given here.

Trifluoroethanol (TFE = CF3CH20H) was 99+% purefrom Aldrich. pH determination in samples containing TFEwas made as described by Nelson and Kallenbach (21).

RESULTSHelix Formation. Peptide helix formation is monitored by

CD. At low temperature (PC) all peptides show spectraindicative of an a-helix; they have the characteristic doubleminima at 222 nm and 208 nm (Fig. 2a). The extent of helixformation is most easily monitored by following the minimumat 222 nm, -[61222. Table 1 lists helix content measured by-[61222 in both 1.0 and 0.01 M NaCl (1PC, pH 7) for thepeptides studied. All peptides show significant helix forma-tion; -[]222 varies from 6,100 to 25,100 cm2*deg-dmol- ,indicating p15-80%o helix content (see estimates below basedon extrapolation from peptides in high TFE concentrations).

Helix formation is an enthalpy-driven process. Unfoldingincreases with temperature in the same manner observed

previously for designed peptides with ion pairs (9) and for theC peptide and its analogs (5-8) (Fig. 2b). The thermal unfold-ing transition is very broad, spanning >70°C. The absence ofa plateau at low temperatures is consistent with the observa-tion that helix formation is only partial under these conditions.

Concentration Dependence of Helix Formation. Studies ofhelix formation as a function of peptide concentration indi-cate that the helix-forming unit is monomeric. Fig. 3a showsthe lack of concentration dependence of -[61222 for peptide3K(I) in helix-forming conditions, 1°C and pH 7, as expectedfor a monomeric species. Fig. 3b illustrates a similar test forconcentration-dependence made throughout the unfoldingtransition zone, using guanidine hydrochloride as a denatur-ant, to ensure that the test is made at the level of stability mostsensitive to changes in peptide concentration. The CD signalis independent of peptide concentration at all denaturantconcentrations. This approach is illustrated in Fig. 3 c and dwith results from the literature for a system in which helicalstability does depend on concentration. The peptide associ-ates into four-helix bundles (15), and helix formation isstrongly dependent on concentration throughout the wholerange of helix stability. In dimeric a-helices that form coiledcoils, no dimer helix formation has been seen with peptidesshorter than 29 residues (16).

Effect of the Number of Charged Residues. The role of thelysines in affecting helix formation was examined by varyingtheir number (three, four, or six) and position within thesequence (Table 1). At neutral pH, the best helix formers are3K(I) and 3K(II); -[1f222 = 25,100 and 24,800 cm2-deg'dmol-1, respectively, at 1°C, 1.0 M NaCl. Adding anotherlysine, as in peptide 4K, results in a small decrease in helixstability. Peptides containing six lysines show, however, asubstantial decrease in helix content.

Effect of the Type of Charged Residue (Lysine and GlutamicAcid). The specific effect of the charged residue was evalu-ated by changing the nature of the charged group frompositively charged lysine to negatively charged glutamate. Atneutral pH a peptide with three glutamic residues shows a

40 60 80(micromolar)

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0 1 2 3 4[GuHCI] (M)

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FIG. 3. Test for dependence of helix forma-tion on peptide concentration for the peptide

d 3K(I) (a and b) and for the peptide a1A designedby DeGrado and coworkers (15) to associateinto a four-helix bundle (c and d). These figuresillustrate by comparison that helix formation inthe alanine-based peptide is monomolecular;data for c and d were taken from ref. 15. (a)Dependence of helicity on peptide concentra-tion for the peptide 3K(I) (0.01 M NaCl, pH 7,1°C). (b) Dependence of helicity on concentra-tion of guanidine hydrochloride at 16.3 juM (0),8.2,uM (+),and 4.1 AM (x) 3K(I) at pH 7 and1°C. (c) Dependence of helicity on peptide con-centration for the peptide ajA. (d) Dependenceof helicity on concentration of guanidine hydro-chloride at several concentrations of ajA: 5 ,uM(0), 10 .tM (+), 17 AM (x), and 28.5 IAM (x).

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similar helix content as either peptide with three lysines.Thus, peptides with either three glutamic or three lysineresidues show unusually stable helix formation.pH Dependence of Helix Formation. The importance of

charge effects on helix content can be evaluated by measur-ing helix formation as a function of side-chain ionization orpH. Fig. 4 shows the pH dependence of -[ 01222 at MC and0.01 M NaCi for three lysine-containing peptides. All threepeptides show qualitatively similar curves independent ofthespecific placement of the lysine residues. The helix is leaststable at neutral pH, and helicity increases in the basic range.The apparent pK indicates that this effect arises from titrationofthe E-amino group of lysine. Peptide association at high pH(>10) has not been studied.

Effect of TFE. TFE, an organic solvent known to promotehelix formation in peptides (21), was used to obtain the valueof -[0]222 for maximal helix formation by each peptide. Helixcontent as a function ofmol% TFE is shown in Fig. 5 for threepeptides containing three, four, or six lysines. At high con-centrations of TFE, all three peptides display the same CDspectrum and - [0]m2 reaches the same maximum value of:32,000 degcm2-dmol-1. This value is reasonable for 100%ohelix formationall peptides shoTFE concentrathe different sptrue differencesfrom sequence-

Unusual Pepiprisingly stableditions, 16-resalanines showformation in a sdesigned peptidacid and lysinepeptides are ccthe stable peptjalanine. Amon,alanine contenthelices than thpeptides with tilysines [3K(I) Echarged glutandominant factoand that alanin

3000a

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FIG. 4. pH tilysine-containingM NaCI at 10C.

0Ela0C0

E0

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Cl

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FIG. 5. Dependence of helicity on TFE concentration for threelysine-containing peptides-3K(I) (e), 4K (A), and 6K(I) (m)-at pH7 and 1(C.

in peptides of this length. Moreover, because no longer surprising, therefore, that in our previous work (9)ow the same value of -[0]2m at sufficiently high peptides with glutamic and lysine pairs spaced three aminoltions, the different values of -[0]222(and also acids apart (i+3; Fig. 1) form moderately stable helicesiectral features) seen at 0%6 TFE must reflect (25-50%o) even though in the (i+3) spacing there is nos in helicity and not just differences resulting evidence of stabilization by salt bridges.-dependent spectral properties. Possible Explanations of the Unusual Helix Stability. The

DISCUSSION most likely explanation for our results is that individualalanine residues have a high helix-forming potential. Theretide Helix Stability. These peptides form sur- are three obvious types of side-chain interactions, however,a-helices. Under optimal helix-forming con- that might provide an alternative explanation to these results,idue peptides containing 3 lysines and 13 but each of these interactions can be eliminated. (i) Lateralas much as 80% helix. Such stable helix association between helices is known to be helix-stabilizing

short peptide has been seen previously only in when it occurs (15, 16). This interaction can be ruled outles stabilized by salt bridges between glutamic because we find convincing evidence that helix formation isfour residues apart (i+4). Although these new)mpositionally quite similar to those, most of monomolecular (see Fig. 3 and Results). (ii) Charged-ides studied here contain a higher content of group-helix dipole interactions are known to stabilize shortg these peptides, helix stability parallels the helices (22, 23). This interaction cannot be the sole explana-t. Peptides with three lysines form more stable tion for stable helix formation for the following reasons.iose with four or six lysines. Comparison of Peptides 3K(I) and 3K(II) contain a positively charged group

at opposite ends of the helix, yet both peptides show sub-

and 3K(II)l shows little effect of substituting stantial helix formation. Moreover, helix stability increasesrate for charged lysine. We conclude that a as the charge is removed from the lysine residues by pHor in these peptides is the high alanine content titration (Fig. 4), whereas charged group-helix dipole inter-e is a strongly helix-favoring amino acid. It is actions must disappear as the charge disappears. Note that

helix stabilization by ion pairs also can be ruled out becausethese peptides contain charged residues of only one chargetype. (iii) Hydrophobic interactions can result from partialburial ofnonpolar surfaces when helix formation occurs. Thisfactor has been discussed often, but experimentally separat-ing it from other factors affecting helix stability is difficult.This subject is considered in a separate section (see below).Comparison with Earlier Work. Unusual helical stability

A for a block of L-alanine residues has been observed in earlierstudies (24, 25) beginning with the pioneering study byGratzer and Doty (24) in 1963. Blocks of D,L-glutamate (24)or D,L-lysine (25) were used to solubilize the alanine blocks.Two explanations have been offered for the perplexing

stability of the alanine helix in these block copolymers. In thelonger block copolymers, self-association of alanine blockswithin one polymer molecule may occur (25). In shorter block

p I I I copolymers, where lateral association between alanine helices2 4 6 8 1 0 1 2 within one polymer is unlikely, a helix-stabilizing hydrophobic

pH interaction has been postulated (25, 26) (see section below).Later Ooi and coworkers (12) studied helix formation by a

itration of helicity as measured by -[012n for three single block of 20 alanine residues solubilized by attachmentg peptides-3K(I) (e), 4K (v), and 6K(I) (v)-in 0.01 to an adjacent block of 20 ionized L-glutamic residues. The

primary aim was to find out whether the helical stability ofthe

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(Ala)20 block depends on whether the (Glu)20 block is at-tached at the N-terminal or C-terminal end of (Ala)20. Astrong position effect was found (12). Their study alsoshowed that the alanine block has a high helical stability. Thealanine block is estimated to show =90% helix formation ifone assumes that only the alanine block is helical at neutralpH. Although a (Glu)20 block would not form an a-helix byitself when ionized, the helix, once formed, could propagatefrom the alanine block into the (Glu)20 block. Therefore, thisunusual stability was not considered surprising because thepeptides contained only L-amino acids and were 41 residueslong, which strongly increases the helical probability.

Role of the Hydrophobic Interaction in Stabilizin Isolateda-Helices. The notion that the hydrophobic interaction maybe an important determinant of a-helix stability has a longhistory [see the 1972 discussions by Fasman and coworkers(27) and by Ptitsyn (4) of their own and earlier work]. Itbecame possible to discuss quantitatively the role of thehydrophobic interaction in protein folding reactions whenLee and Richards (28) developed an accurate algorithm forcomputing H20-accessible surface area and Chothia (29)correlated transfer free energies with nonpolar accessiblesurface area for amino acid side chains. Then, assuming thatthe same correlation applies to folding reactions, one cancompute the free energy change corresponding to the burialof hydrophobic groups upon folding.

Richards and Richmond (30) give data for this decrease innonpolar accessible surface area when a given amino acidresidue folds into an a-helix, in which the surroundingresidues are either alanine or the sequences found in thehelices of sperm whale myoglobin. They find that a decreasein nonpolar accessible surface area always accompaniesa-helix formation, that the typical change in Gibbs freeenergy is modest [c0.5 kCal per residue (1 Cal = 4.184J) foramino acids with small side chains], and that the change doesnot vary strongly from one residue to the next within a givensize class. The change found for alanine is not exceptional.Moreover, the difference in the value obtained using either analanine helix as background or the average of the myoglobinhelices is small. The explanation given earlier (26) of theunusual helical stability noted in polymers containing blocksofalanine is based on a specific hydrophobic contact betweenthe a carbon of residue i and the ( carbon of residue i+3.Such an interaction would not be specific for alanine butwould be allowed for all residues containing a P carbon andshould also occur between alanine and the host residue(hydroxypropyl-L-glutamine) used in the host-guest study ofalanine (11) (see the following section).

Consequently, the probable conclusion from our study isthat individual alanine residues have a high intrinsic helicalpotential. Whether alanine is exceptional in this respect orwhether other nonpolar residues also have high helical poten-tials remains to be determined.Comparison with Host-Guest Results. The value of s for

alanine given by the host-guest method is 1.08 at 0°C (11),whereas a preliminary analysis of our results by the Lifson-Roig theory (31), in collaboration with J. A. Schellman, givess (0WC) :2, or a 2-fold difference. (This preliminary analysisuses the host-guest value of s for lysine, which is 0.94 at20°C.) In 1966 Berger and coworkers (10) studied the helix-forming properties ofHBLG (hydroxybutyl-L-giutamine) andHPLG (hydroxypropyl-L-glutamine), the host residues ofhost-guest studies, and HELG (hydroxyethyl-L-glutamine).They found that HBLG forms a moderately stable helix inH20, but that HELG, like poly(L-glutamine) itself, does notform a helix in H20. HPLG forms a helix of intermediatestability. All three derivatives-HBLG, HPLG, andHELG-show stable helix formation in organic solvents suchas methanol. Berger and coworkers (10) concluded that theHBLG helix is stabilized by hydrophobic interactions be-

tween adjacent hydroxbutyl side chains. Accepting theirexplanation, we suggest that in the copolymer of alanine andHPLG used in host-guest studies of alanine (11), the shortalanine side chain is unable to take part in the hydrophobicinteraction that involves the hydroxypropyl moiety of theHPLG residue. This failure to participate in a helix-stabilizinginteraction among host residues thus appears as a helix-destabilizing factor in the helical potential ofalanine found bythe host-guest method. The result is that the helical potentialof alanine is underestimated.

It is important now to test this and other possible expla-nations for the difference between our results and those ofScheraga and coworkers (11); they note internal consistencybetween their host-guest values and the earlier block copoly-mer results by Ingwall et al. (25). The exact basis for thiscontradictory behavior is important for understanding thea-helix and remains a goal for the future.

We thank Daniel Horowitz for his valuable contribution to theinitial phase of this study, Dr. Harold Scheraga for his discussion ofthe difference between our results and the host-guest results foralanine, and Fred Hughson and Alan Sachs for critical review of themanuscript. Dr. John Schellman made available his computer pro-gram for analyzing our results in terms ofthe Lifson-Roig theory andmade valuable suggestions about interpretation. S.M. is a predoc-toral fellow of the National Institutes of Health Medical ScientistTraining Program (GM07365). Mass spectra were provided by theUniversity of California (San Francisco) Mass Spectrometry Re-source, supported by National Institutes of Health Grant RR 01614.This research was supported by National Science Foundation GrantDMB 881-8398.

1. Creighton, T. E. (1983) Proteins (Freeman, New York).2. Shoemaker, K. R., Kim, P. S., Brems, D. N., Marqusee, S., York, E. J.,

Chaiken, I. M., Stewart, J. M. & Baldwin, R. L. (1985) Proc. Nati.Acad. Sci. USA 82, 2349-2353.

3. Sueki, M., Lee, S., Powers, S. P., Denton, J. B., Konishi, Y. &Scheraga, H. A. (1984) Macromolecules 17, 148-155.

4. Ptitsyn, 0. B. (1972) Pure Appl. Chem. 31, 227-244.5. Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476.6. Bierzynski, A., Kim, P. S. & Baldwin, R. L. (1982) Proc. Nati. Acad.

Sci. USA 79, 2470-2474.7. Rico, M., Nieto, J. L., Santoro, J., Bermejo, F. J., Herranz, J. &

Gallego, E. (1983) FEBS Lett. 162, 314-319.8. Kim, P. S. & Baldwin, R. L. (1984) Nature (London) 307, 329-334.9. Marqusee, S. & Baldwin, R. L. (1987) Proc. Nati. Acad. Sci. USA 84,

8898-8902.10. Lotan, N., Yaron, A. & Berger, A. (1966) Biopolymers 4, 365-368.11. Platzer, K. E. B., Ananthanarayanan, V. S., Andreatta, R. H. & Scher-

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Proc. Natl. Acad Sci. USA 86 (1989)

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