Solution Properties of Synthetic Polypeptides 453
Vol. 7, No. 4, July-August 1974
X = 213 mp 258 x X = 150-180 m p -572 X
> RZI3> 223 x lo-' < Rl;o-180< -561 X
are very difficult to compare with the results obtained in
W E . They do not permit one to associate a definite change in the n-T* rotatory strength and the breaking of the helical structure.
T-T*
splitting to
Conclusion While limited by the absence of a sufficiently transparent nonhelicogenic solvent, the analysis of ORD and CD performed in this study leads to two interesting conclusions: (1) the n-T* rotatory strength in the helical polymer is a t least twice as intense as in model compounds; (2) the T-T* transition is split into two bands. Since we show in a subsequent articles that the geometry of the PHB helix in solution can be precisely known from nmr data using [shift reagents, we hope that these results will help to test the applicability of the theory relating n--K* rotatory strengths to the conformation.10 As far as T-T* transitions are concerned, the sign of the splitting
could be tested by linear dichroic measurements on oriented films since it is known t h a t the more displaced band is always polarized parallel to the helix axis. An effort is needed to find nonhelicogenic solvents with sufficient transparency for the observation of the n-T* CD band and Cotton effect. References a n d Notes (1) J. Cornibert, R. Marchessault, H. Benoit, and G. Weill. Macromolecules, 3,741 (1970). (2) R. H. Marchessault, K., Okamura, and C. J. Su, Macromolecuies, 3, 735 (1970). ( 3 ) W. Moffitt, D. Fitts, and J. C. Kirkwood, R o c . Nat. Acad. Sei. C: S.. 43, 723 (1957). (4) H. T. Clarke, J. Org. Chem., 24,1610 (1989). ( 5 ) J. Olsen, J. M. Merrick, and I. J. Goldstein, Biochemistry, 1, 453 (1965). (6) A. Moscowitz in "Optical Rotatory Dispersion," C. Djerassi, Ed., McGraw-Hill, Yew York, Y. Y., 1960, p 160. (7) K. K . Yamaoka, Biopolymers, 2, 219 (1964). (8) I. Tinoco,J. Amer. Chem. Soc., 86,297 (1964). (9) J. Delsarte and G. Weill, Macromolecules, in press. (10) P. M. Bayley, E. S. Sielsen, and S. A. Schellmann, J , Phys. C h e m , 73,228 (1969).
Solution Properties of Synthetic Polypeptides. Assignment of the Conformation of Poly(L-tyrosine) in Water and in Ethanol-Water Solutions E. Peggion,* A. Cosani, and M. Terbojevich Institute of Organic Chemistry, University of Padoua, 35100 Padova, Italy. Received November 13, 1973
ABSTRACT: The CD absorption properties of poly(L-tyrosine), (L-Tyr),, poly(L-lysine). (L-Lysj,, and of random copolymers of tyrosine and lysine have been investigated in water and in water-ethanol mixtures. In aqueous solution at pH 10.9 and 11.2 a gradual variation of the CD properties was observed on increasing the tyrosine content in the peptide chain from 0 to 87.5% (on a molar basis). These data indicate that the entire copolymer series at pH 10.9 and 11.2 assume the same conformation as pure ( L - L Y S )namely, ~, a right-handed a helix. On going from 87.5% tyrosine to 100% tyrosine in the chain there is a sharp change of the CD properties, corresponding to a conformational transition. Under our experimental conditions pure (L-Tyr), a t p H 5 11.2 is not in the righthanded a-helical form but in the R structure. Evidence is presented that the t3 structure is very unstable. Comparison with literature data lead to the conclusion that ordered (L-Tyr), can assume either the R form or the LI helix depending on the experimental conditions. Similar copolymer studies carried out in water-ethanol mixture containing 92% ethanol show that the entire copolymer series and (L-Tyr)n as well assume the right-handed N helical form. 'The differences between the CD properties of the tyrosine-rich copolymers in the two solvent systems are interpreted in terms of solvent effects.
In spite of many investigations carried out by different research groups in different laboratories, the conformation in solution of poly(L-tyrosine), (L-Tyr),, has not been securely and unequivocally established. Fasman and coworkers1-3 first reported t h a t the polymer undergoes a pH-induced conformational transition from an ordered form to a random coil. According to these authors the ordered form is that of a right-handed a helix, which can be obtained only following a detailed procedure which involves a very slow lowering of the p H from alkaline conditions to p H 11.2. The CD pattern of helical (L-Tyr), in aqueous solution is chlaracterized by a positive band a t 245 nm followed by a negative band a t 224 nm and again by a strong positive band a t 200 nm. Substantially the same spectrum has been recently reported by Quadrifoglio e t al.,* with one significant difference. The negative band is located a t 230 nm and not a t 224 nm. Soon after the papers of Fasman, theoretical calculations carried out by
Pao et a1.5 and by Scheraga and coworkers6-7 gave further support to the conclusion that (L-Tyr)n under proper pH condition assumes a right-handed a-helical form. However, these conclusions have been seriously questioned by subsequent work carried out by different authors. Applequist and Mahr8 by dielectric dispersion techniques in dilute quinoline solution assigned the left-handed e-helical conformation to (L-Tyr),. Patrone e t al.,9 using ir and ultracentrifugation techniques concluded that the polymer assumes the antiparallel (3 conformation a t pH 5 11.2. Essentially the same conclusion was drawn by Senior et ~ 1 . 1 0in their careful work based on light-scattering and potentiometric titration studies. Conio e t al.ll carried out ir measurements in water-ethanol mixtures and concluded that (L-Tyr), assumes the a-helical form in ethanolrich solvents, the sense of the helix obviously being undetermined. Very recently, Friedman and Ts'o12 found for (L-Tyr),
454
Peggion, Cosani, Terbojevich
a t pH 11.2 a CD pattern substantially identical with that reported by Fasman1-2 and Quadrifoglio et u L . , ~ except for a small blue shift of the negative band, which is located a t 227 nm. At p H 10.6 the same CD pattern as that reported by Shiraki and Imahory13 in methanol, by , ~ Engel et al.15 in triDamle,l4 Quadrifoglio et ~ l . and methyl phosphate, was found. The spectrum is characterized by a positive band a t 230 nm, followed by a very weak negative band a t 217 nm, and by a very large positive band at 200 nm. Largely because this pattern is consistent with theoretical calculations of Chen and Woody,16 Friedman assumed that (L-TYr)n in water a t pH 10.6 in methanol and in T M P assumes the right-handed a-helical form, and that the polypeptide chain retains its helical structure a t pH 11.2, with a considerable increase of the degree of ionization of the side chains. It is clear from all this controversial story that the assignement of the conformation in solution of (L-Tyr)n is far from being securely and unambiguously assigned. The optical rotatory properties of the homopolymer are of little help in this case because of strong overlapping contributions to the optical activity from side-chain and mainchain chromophores. On the other hand, all theoretical calculations which predict the right-handed a helix as the most stable conformation do not take into account the presence of solvent, which can change everything. Finally, it should be noted that the theoretical work of Pao et a1.5 leads to an ORD pattern for the right-handed a-helical form, which is in agreement with the experimental results of Fasman and c o ~ o r k e r s . l -But ~ according to a number of workers,8-12 the ORD pattern of (L-Tyr), reported by Fasman is not that of an a helix. In the present paper we will present decisive evidence that (L-TYr), assumes the right-handed a-helical form in ethanol-water (92% ethanol) and in trimethyl phosphate, and that the polymer conformation in aqueous solution a t pH 5 11.2 can be either a right-handed a helix or a fi structure, depending on the experimental conditions. Our contribution involves the synthesis and the study of the CD properties of (L-Tyr), and of random copolymers of L-lysine and L-tyrosine containing various proportions of aromatic residues in the chain.
Macromolecules Table I Elemental Analysis on Random Copolymers of 0-Carbobenzoxy-L-tyrosine and Ne-Carbobenzoxy-L-lysine Anal.
mer
Tyr Content (mol%)
1 2 3 4 5 6 7
12.5 25.0 37.5 50.0 62.5 75.0 87.5
Copoly-
Calcd (%) C
H
F o u n d (%)
N
64.64 6.63 9.93 65.21 6.41 9.19 65.78 6.18 8.44 66.35 5.96 7.70 66.93 5.73 6.95 67.50 5.50 6.20 68.07 5.28 5.45
C 63.69 64.57 64.89 66.92 66.20 66.93 66.77
H
N
6.24 9.49 6.08 8.79 5.86 8.02 6.04 7.82 5147 6.64 5.35 5.82 5.13 5.13
poured into ethyl ether. The precipitated polymer was recovered by filtration, washed with ethyl ether and dried under vacuum a t 50". The reduced viscosity (vsp/c) of this sample in CHClzCOOH was 2.04 dl/g (c 0.2 g/dl). Poly(L-lysine hydrochloride) was prepared from the remaining portion of the poly(Nkarbobenzoxy-L-lysine)solution by treatment with anhydrous HBr according to the literature.21 The intrinsic viscosity of the polymer was [qJ = 1.00 a t 25" in 0.2 M NaC1. Poly(0-carbobenzoxy-L-tyrosine)was prepared by polymerization of the corresponding N-carboxyanhydride in anhydrous dioxane with triethylamine as the initiator. The molar ratio monomer to initiator was 30. Also in this case a small portion of polymer was recovered from the solution (by pouring the solution into petroleum ether). The reduced viscosity of this sample was 1.53 dl/g in CHClzCOOH a t 25" (c 1.53 dl/g). Poly(L-tyrosine)(L-Tyr), was obtained from the remaining solution of poly( 0-carbobenzoxy-L-tyrosine) by removing the carbobenzoxy-protecting group with anhydrous HBr according to the procedure described by Fasman.l In order to prevent the lightinduced decomposition of HBr and to avoid possible bromination of the phenol ring the reaction was carried out in the dark. The polymer precipitated during the reaction was recovered by filtraM HCI, tion, dissolved in 1 N NaOH, and dialyzed against using an A 4465-A 2 dialyzing tubing (A. Thomas Co., Philadelphia, Pa.), which retains materials with mol wt 12,000 or higher. The product was finally recovered by lyophilization and was in the form of a perfectly white powder. The intrinsic viscosity was [ v ] = 2.15 dl/g in 0.2 M N a C l at p H 12. Random copolymers, of Ne-carbobenzoxy-L-lysinea n d O-carbobenzoxy-L-tyrosine were prepared by copolymerization a t room temperature of known amounts of the corresponding N-carboxyanhydrides in anhydrous dioxane using triethylamine as the Experimental Section initiator. In all copolymerization experiments the total monomer Materials. Reagent grade dioxane (Carlo Erba RPj was dried concentration was 0.1 mol/l. and the monomer to initiator molar over potassium anthracenate according to the literature'? and ratio was 30. A t the end of the reaction (checked by irj samples of distilled immediately before use. The water content was less than each copolymer were isolated by pouring small portions of the co0.002% (by weight). Reagent grade petroleum ether (bp 40-70", polymers solutions into ethyl ether. Each copolymer was recovCarlo Erba RP) was refluxed over sodium wire and then fractionered by filtration, washed, and dried under vacuum a t 50". The ally distilled. Chloroform and ethyl acetate (Merck Puriss. j were analytical data on the copolymers so obtained are summarized in dried over potassium carbonate and then fractionally distilled. Table I. Acetone (Merck Puriss.) was treated with Drierite and then disFrom the remaining portions of the blocked copolymers solutilled. Reagent grade triethylamine (Fluka Puriss.) was refluxed tions diluted with equal amounts of CHC13, the corresponding deover pellets of KOH; the supernatant liquid was recovered by deblocked lysine-tyrosine copolymers were obtained by reaction cantation and then distilled from potassium metal. Ethanol with anhydrous HBr, following exactly the same procedure as for (Merck Spectrograde), ethyl ether (Carlo Erba RP), and dichlo(L-LysIn. roacetic acid (Carlo Erba RP) have been used without further puAfter recovering by filtration, the lysine-rich copolymers 1-2-3 rification. were dissolved in 1 N HC1, being insoluble in alkaline conditions. Monomers. Ne-Carbobenzoxy-L-lysine-N-carboxyanhydride Copolymer 4 containing equal molar amounts of tyrosine and lyswas prepared from NC-carbobenzoxy-L-lysinelsby reaction with ine residues is soluble both in acid and alkaline aqueous solution, phosgene in anhydrous dioxane according to the 1 i t e r a t ~ r e .The l~ while the tyrosine-rich copolymers 5-6-7 are soluble only in alkacrude material was recrystallized several times from ethyl aceline conditions. The aqueous solution of each copolymer (basic or tate-petroleum ether, m p 100" (lit.19 100"). 0-Carbobenzoxy-Lacid depending on the composition of the copolymers) was dityrosine-N-carboxyanhydride was prepared according to the literalyzed against 0.01 N HC1. The samples were then recovered by atureZ0 by reaction of 0-carbobenzoxytyrosine with phosgene. lyophilization, and were in the form of perfectly white powders, After several recrystallizations from ethyl acetate-petroleum which have been characterized by amino acid analysis. The ether, a pure product was obtained with m p 106" (lit. 101"). amino acid analysis was carried out after complete hydrolysis of Polymers and Copolymers. Poly(Nc-carbobenzoxy-L-lysine) copolymer samples in 6 N HC1 at 110" for 24 hr, in the presence of was obtained by polymerization of the corresponding N-carboxytraces of The amino acid analysis data on the deblocked anhydride in dioxane using triethylamine as the initiator. (The copolymers are reported in Table 11. monomer to initiator molar ratio was 50.) At the end of the reacPreparation of Polymers a n d Copolymers Solutions for CD tion, checked by ir, a small portion of the polymer solution was Measurements. The different solubility properties of the homo-
Solution Properties of Synthetic Polypeptides 455
Vol. 7, NO.4, July-August 1974 Table I1 Amino Acid Analyt3isa on Random Copolymers of L-Tyrosine and L-Lysine 2
Tyrosine Content (mol % )
Copolymer
Calcd from the Re1 Amounts of M'onomers in the Polymerizn Mix.
Found
1 2 3 4
12.5 25 .o 37.5 50.0
12.8 25.3 40.5 49.7
Amino acid analyses were not carried out on copolymers 5-7 because of the unc'omplete solubilization of the samples Q
during the hydrolysis procedure. The amount of tyrosine was therefore assumed identical with the theoretical value. This assumption is justified from the data of Table I.
1
7
-
0
X
2%
-1
polymers and of the copolymers require different procedures t o obtain solutions at the desired pH conditions. Aqueous solutions of (L-Tyr)n and of the tyrosine-rich copolymers 5-6-7 were prepared by suspending ca. 10 mg of each sample in 20 ml of water and then adding 1 N KOH to complete dissolution which generally occurred at pH N 12. All solutions were then back-titrated very 'jlowly to pH 10.9 with 1 N HC1 under vigorous stirring. (L-Lysj, and the lysine-rich copolymers 1-2-3 were directly dissolved in water ( - 10 mg in 20 ml) and directly titrated to pH 10.9 with 1 N KOH. Unfortunately, following either the back or the direct titration procedure, copolymer 4 was not soluble at pH 10.9 even at concentrations lower than 0.05 mg/ml. It was soluble at pH 11.2 following the back-titration procedure from pH 12. Polymers and copolymers solution in water-ethanol mixtures were prepared according to the following method. (L-Tyr)nand copolymers 5-6-7 ( - 10 mg of each sample) were suspended in 2 ml of water and 1 N KOH was added to complete dissolution. Then ethanol was added under stirring until reaching 92% ethanol (v/v) in the solvent mixture. Slightly opalescent solutions were obtained. On lowering the apparent pH to 5 , all opalescence disappeared and perfectly clear solutions were obtained on which CD measurements were carried out. (L-Lysjn and copolymers 12-3-4 ( - 10 mg of each sample) were dissolved in 2 ml of water and the proper amounts of ethanol were added under stirring. Clear solutions were obtained and the apparent pH was adjusted to a value of 5. Measurements. CD measurements have been carried out at room temperature, using a Cary 60 spectropolarimeter equipped with a 6002 CD accessory unit. Fused quartz cells (0.5, 1.0, and 10 mm) with Suprasil windows were used. pH measurements were performed with a Metrohm Model E 388 precision potentiometer and using either Metrohm UX combined glass electrodes or Beckman glass and Kalomel electrodes.
Results and Discussioin The CD patterns of pure poly(L-tyrosine) a t p H > 12 and at pH 10.9, in absence of added salts are presented in Figure 1. On the same figure the CD results obtained by Fasman,1,2 and by Qundrifoglio4 are reported for comparison. The spectrum at p H 10.9 has been obtained following exactly the back-titration procedure described by Fasman and coworkers.l.2 While there is a qualitative agreement among the results obtained by different workers as far as the shape of the spectrum is concerned, it appears clearly t h a t the negative band a t 230 n m found by us is much stronger than that reported by others. The molar ellipticity a t 230 n m is in fact -21,300, against -5000 and -9000 reported by Friedman12 and Q ~ a d r i f o g l i o . ~ On carefully lowering the p H from 10.9 to 10.6 we were not able to obtain the CD spectrum obtained by Friedman, in spite of having followed exactly the same procedure. We believe t h a t this is due t o the different sample of poly(L-tyrosine) used by different research groups. In the experience of many laboratories, including ours,
-2
-3
I
2
I
220
I
I
260
240
I
I
280
, 300
A (nm)
Figure 1. CD spectra of (L-Tyr), at pH 10.9 (curve la). at pH 11.2 (curve lbj and pH 12 (curve 4). The CD spectra obtained by Fasman in 0.2 M NaCl at pH 11.2 (curve 2) and Quadrifoglio in 0.1 M NaC104 at pH 10.8 (curve 3) are reported for comparison. poly(a-amino acids), when purchased by chemical companies, have generally to be carefully checked for purity and molecular weight (which is very often greatly different from that claimed by the vendors) .23 In the case of poly(L-tyrosine) one often obtains yellow samples, due probably to bromination of the phenol ring during the deblocking reaction. This fact might of course affect the optical properties of the polymer. While there is a general agreement in assigning the random-coil conformation to (L-Tyr), in aqueous solution a t p H > 12, no interpretation of the CD pattern in terms of conformation can be made of the ordered form of the polymer at p H 5 10.9, because of overlapping contributions from the side-chain and main-chain chromophores. Following the same approach of previous research on the conformation of aromatic poly(a-amino a ~ i d s ) , ~ ~ - ~ 8 the CD properties in aqueous solution of random copolymers containing variable amounts of L-tyrosine and L-lysine have been investigated. At p H 10.9, in absence of added salts, both (L-Tyr), and (L-LYS),are in a n ordered conformation, which is a right-handed N helix in the case of ( L - L ~ s )By ~ . observing the perturbations on the CD pattern of pure ( L - L ~ s ) ,induced by increasing proportions of tyrosine residues in the chain, useful information can be obtained concerning the ordered form of (L-Tyr),. The results of such investigations are shown in Figure 2. It is evident t h a t a relatively small amount of lysine in the (LT Y ~chain ) ~ ( 13 mol %) causes the complete collapse of the ordered form. A new CD spectrum is obtained (see copolymer 7 in N
456 Peggion, Cosani, Terbojevich
Macromolecules
/
cop
'
pH lo
i
1
i
i
i
i
1
i
i 0
E!c
t
x
'9
rn
X
a
(L-Tyr),
-1
I
pH 10.6
/
i \
-2
-3
200
220
240
260
280
(nm)
Figure 2. (a) CD spectra of (L-TyrIn, (L-LYS),, and of some of the random copolymers of lysine and tyrosine in aqueous solution a t pH 10.9, in absence of added salts. The numbers on the curves refer to the number of the copolymers (Tables I and 11). (b) CS spectra of copolymer 7 a t pH 10.9. The CD spectrum of (L-Tyr)n obtained a t p H 10.6 by Friedman and of (L-Tyr)n a t p H 12 are also reported for comparison.
Figure 2 ) which is not that of the coiled form of ionized (L-Tyr)n. The CD pattern (Figure 2b) is characterized by a shoulder a t 245 nm followed by a maximum a t 233 nm ([e]= 9000), and a minimum a t 217 nm ([e]= -2800). Finally there is a strong positive band a t about 202 nm. Except for the shoulder a t 245 nm and for the band intensities the CD pattern of copolymer 7 is qualitatively similar in shape to that found by Friedman12 in water a t p H 10.6. On further increasing the lysine content of the copolymers there is a gradual variation of the CD pattern from copolymer 7 to pure (L-LYS),. This is even more evident from Figure 3, where the molar ellipticity values at 230 nm are reported as a function of the copolymer composition. From these data there is strong evidence for a conformational change on decreasing the tyrosine molar content in the peptide chain from 100 to 87.5%, while a further decrease of the tyrosine content seems to be without any effect on the polypeptide conformation. The same results have been obtained on performing the measurements in water a t pH 11.2 where all the copolymers are soluble (Figure 3). The conclusion therefore follows that between pH 10.9 and 11.2 the conformation of the entire copolymer series is identical with that of pure (L-LYS),, namely, a righthanded a helix, and that pure (L-Tyr), under the same pH conditions assumes a different conformation. This conclusion is consistent with the results obtained in water by various authors9~~0 using different experimental tech-
niques, who assigned the @ structure for (L-Tyr), a t pH 10.9. By comparing the CD data on copolymer 7 (which contains the highest amount of tyrosine residues) with those obtained by Friedman on (L-Tyr), a t pH 10.6 (Figure 2b), it follows that Friedman was able to obtain the polymer in the a-helical form. The intensities of the CD band a t 230 nm plotted us. the copolymer composition extrapolate well to the [e] value reported by Friedman for pure (L-Tyr), (Figure 3). With our polymer sample we were unable to reproduce the results of Friedman a t p H 1.06, but it is probable that small variations of temperature and salts content are sufficient to induce the @-helix transition of (L-Tyr)n in water. The instability of the p form of ( L - T V ) ~is evident from our CD studies on the lysine-tyrosine random copolymers. It is just sufficient to introduce 12% lysine residues to have the complete transition to the right-handed a-helical form. For the nonlinear variation of the CD pattern on going from pure ( L - L ~ sto ) ~ copolymer 7 we can give the same explanation as in the case of tryptophan- and phenylalanine-containing cop0lymers,~4-26 that is the progressive introduction into the peptide chain of aromatic residues containing optically active side chains allows electronic interactions among these chromophores, whose contributions to the optical activity are not simply additive. In the specific case of (L-Tyr)n the existence of interactions among phenol groups has been established by other investigators .29
Solution Properties of Synthetic Polypeptides 457
Vol. 7, NO.4 , July-August 1974
EtOH
31 2t
-31
10
20
30
40
50 60 70 tyrosine (mole %)
00
90
Figure 3. Molar ellipticity values [ B ] at 230 nm (from Figure Pa) reported as a function of the copolymer composition. The extrapolated value on the right portion of the curve corresponds to the molar ellipticity a t 230 n m reported by Friedman for (L-Tyr), a t p H 10.6. I
CD studies on random lysine-tyrosine copolymers allowed the assignment of the conformation of (L-Tyr), in water-ethanol solvent mixtures. In this solvent mixture the conformational properties of the polymer have been previously investigated by Conio e t al.ll using ORD, ir, and potentiometric techniques. These authors observed that in solvent mixtures containing more than 45% ethanol (L-Tyr), a t low degrees of ionization assumes an ordered form, which was interpreted as an cy helix on the basis of infrared measurements. Obviously infrared measurements do not allow us to assign the sense of the helix of the polymer. The results of CD measurements carried out on (L-Tyr), and on random tyrosine-lysine copolymers in water-ethano1 mixtures are shown in Figures 4-7. As described in the Experimental Section, in all measurements the apparent pH was ca. 5 . In 92% ethanol the CD pattern (Figure 4) is qualitatively consistent with that reported by Shiraki and Imahory in methanol,I3 by Friedma+ in water a t pH 10.6, and by a number of workers in TMP.4J4J5The spectrum is characterized by a positive band a t 230 nm, followed by a small negative band a t 217 nm and by a very strong positive band a t 200 nm. The intensities of these bands differ considerably from those reported in the literature, and this is probably due to solvent effects, since the spectra reported by different authors have been recorded in different solvent media. The importance of solvent effects is evident from Figure 5 , where the molar ellipticity values at 230 nm are reported as a function of the solvent composition. Above 30% ethanol, where the conformational transition is essentially complete the CD pattern changes linearly with the ethanol content, and this behavior is clearly due to solvent effects. The CD spectra of lysine-tyrosine random copolymers in 92% ethanol are shown in Figure 6. Starting from pure (L-LYS),, on increasing the tyrosine content of the copolymers there is a gradual monothonic variation of the CD pattern. This behavior is more evident from Figure 7, where the intensity of the 230-nm band is reported as a function of the copolynier composition. The graph shows clearly that no conformational transition takes place on increasing the tyrosine content of the peptide chains. Since the conformation of protonated (L-LYS),in 92% eth-
1
I
I
200
I
240
220
1
1
1
260
. ,__,
A (nm)
Figure 4. CD pattern of (L-Tyr), in 92% ethanol a t the apparent pH Of 5.
5l
4
3P I
0
-
2-
X
0
E 1-
3
-v 0-
-2
I
1
I
1
10
20
30
40 50 60 E t O H (V/V)
*/.
70
80
90
11 0
Figure 5. Molar ellipticity values a t 230 nm of (L-Tyr), in waterethanol mixtures, in function of the ethanol content.
anol is that of a right-handed a helix,3O it can therefore be concluded that (L-Tyr), in this solvent also assumes the same conformation. In this case also, electronic interactions among the aromatic side-chains chromophores account for the nonlinear variation of the CD pattern as a function of the copolymer composition. It is pertinent at this point to compare the CD spectra of the tyrosine-lysine copolymers observed in water a t pH 10.9 and in. ethanol-water solution containing 92% ethanol. According to the above conclusions, the entire series of copolymers in the two solvent systems assume the right-handed a-helical conformation. We note first that the CD spectra of the tyrosine-rich copolymers at pH 10.9 differ from those obtained in ethanol-water mixtures mainly because of the
458 Peggion, Cosani, Terbojevich
Macromolecules
4
3
1
i
1
-31
0
FC
I
1
,
10
20
30
1
1
I
I
40 50 60 70 Tyrosine (mole X )
1
80
90
1
X
Figure 7. Molar ellipticity values a t 230 nm in 92% ethanol reported as a function of the copolymer composition (from Figure 6).
‘ I
u
-1
-2
-3 (L-Tyr ), in 26.7%EtC
~
200
220
240
260
Figure 6. CD spectra of (L-Tyr)n, (L-LYS),, and of random copolymers of lysine and tyrosine in 92% ethanol. In all measurements the apparent p H was 5.
presence of a pronounced shoulder in the region of 250 nm and because of much lower intensities of the bands near to 230 and 200 nm. These differences should be reasonably explained in terms of solvent effects. In fact, if the ethanol content is reduced to 27% one observes that the CD spectrum of (L-Tyr)n,which is still in the a-helical conformation, becomes substantially similar to that of copolymer 7 in water a t pH 10.9 (Figure 8). In this case, the slightly lower intensity of the CD bands of the copolymer is due to the lower tyrosine content. Figure 8 shows also that the CD pattern of copolymer 7 in water is pH dependent; the shoulder a t 245 nm diminishes on lowering the pH from 11.2 to 10.9. This fact suggests that part of the differences in the CD properties of the tyrosine-rich copolymers in aqueous and aqueousalcoholic solution is due to different degrees of ionization of the tyrosine side chains. In fact, for solubility reasons the CD measurements in the two solvent systems were carried out at different pH values. In 92% ethanol a t pHapr, ca. 5 the tyrosine groups can be assumed completely protonated, while in water at pH 10.9, a fraction of tyrosine residues is still ionized. Conclusions The results presented in this paper give rather conclusive evidence that the ordered form of (L-Tyr), in aqueous
190
210
250
230
270
(nm)
Figure 8. CD spectra of copolymer 7 a t pH 10.9, a t 11.2 and of (L-Tyr)na t pH,,, 5 in 26.7% ethanol.
solution a t pH 11.2 and 10.9 is not a right-handed a helix. These findings are consistent with the conclusion of Senior et al.1° and of Patrone et al.,$ who suggested the p conformation on the basis of ir absorption measurements. Furthermore, evidence was obtained that the @ structure of (L-TV), is rather unstable; the introduction of a few lysine residues causes a conformational transition from a /3 form to an a helix. The instability of the /3 form is confirmed by the data of Friedman and Ts’o who obtained the a-helical form at pH 10.6. The right-handed a-helical conformation in ethanol-water solvent mixtures containing more than 30% ethanol has been definitly confirmed on the basis of the gradual variation of the CD pattern of random copolymers lysine-tyrosine.
Molecular Theory of the Helix-Coil Transition in Poly(amino acids) 459
Vol. 7, No. 4, July-August 1974
Acknowledgment. The authors thank Professor E. Scoffone for the stimulating discussions during this work, and Mr. Silvio Da Rin Fioretto and Miss Clara Benvegnu’ for their skilful1 technical assistence. The work has been carried out with the financial support of CNR. References and Notes G . D. Fasman, E. Bodenheimer, and C. Londblow, Biochemistr), 3,
1665 ( 1964).
S.Beychok and G . D. Fasman. Biochemistry, 3,1675 (1964). G . D. Fasman, Nature (London),193,681(1962). F. Quadrifoglio, A. Jus and V. Crescenzi, Makromol. Chem., 136, 241
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Molecular Theory of the Helix-Coil Transition in Poly(amino acids). IV. Evaluation and Analysis of s for Poly(L-valine) in the Absence and Presence of Water1 Mitiko
F. Theodore Hesselink,2bNobuhiro GO,2cand Harold A. Scheraga*
Department of Chemistry, Cornell Uniuersity, Ithaca, New York 14860. Received December 5,1973
ABSTRACT: The Zimm-Bragg parameter s for the helix-coil transition in poly(L-valine) in water was evaluated from the intramolecular interaction energies taking into account two kinds of solvent effects, uiz.,the change in hydration around the side-chain methyl groups (including hydrophobic bonding) which accompanies the conformational change and the binding of water molecules to the free NH and CO groups of residues in the coil state. The entropy loss f.rom the restricted rotational freedom of the side chain in the a helix (in the presence and absence of water) and the binding of water molecules to the free NH and CO groups destabilize the a helix of poly(L-valine); however, the change in hydration around the side-chain methyl groups stabilizes the a helix over the random coil. The s us. temperature curve of poly(L-valine) in water shows a maximum around 50’ which originates from the change in hydration around the side-chain methyl groups. Also, a method for including further interactions beyond those in the random-coil form of the dipeptide is proposed, and the effective range of these additional interactions under experimental conditions is estimated.
In the previous papers3-j of this series, we reported the general formulation of the molecular theory of the helixcoil transition in poly(amino acids)3 and the evaluation of the Zimm-Bragg parameters6 s and u for polyglycine and poly(L-alanine) in the absence and presence of ~ a t e r . ~In, j this paper, we extend the computations to obtain the values of s for poly(L-v,aline) in the absence and presence of water. The evaluation of u for poly(L-valine) was not carried out because of the large amount of computer time required. These computaltions provide a theoretical basis for understanding the behavior of s and u (which are also being determined experimentally for the naturally occurring amino acids using random copolymers7) and for computing the tendency of each amino acid to form or disrupt the 0helical conformation in proteins.s Since poly( L-valine) has bulkier side chains than poly@-alanine), the nonbonded and hydrophobic interactions between the side chains would be expected to play an important role. In poly(L-ala-
nine), these interactions between the side chains contribute neg1igibly.j The formulation and the notation of the molecular quantities follow the previous paper^^-^ except that the recently adopted standard nomenclature conventiong is used for the description of the conformations in the present paper. Only the parameter set A4 (with D = 4) is used for the calculations in the present paper, since this parameter set is more reasonable than the other^.^ Also, only the right-handed CY helix is considered here, and not the left-handed one.
I. Energy Minimization The conformational energy was minimized with respect to the dihedral angles 4L,GL,and xcl, with xL2s1and xL2,2 held fixed at 180’ in both the helical and coil states. (Hereafter, xL1will be denoted simply as xc.) A. Coil State. In paper I,3 the energy of the coil state was taken as a sum of interaction energies F(l)(@c),
