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Conformation of serine polypeptides
J. Mol. Biol. (1968) 36, 355-369
Conformation of Serine Polypeptides Optical Rotatory Dispersion and Infrared Studies NANCY M. TOONEYt
AND GERALD D. FASMAN
Graduate Department of Biochemistry, Brandeis University Waltham, .itfa88achu8ett8 02154, U.S.A. (Received 12 February 1968, and in revised form 22 May 1968) The infrared and optical rotatory dispersion spectra of L-serine polypeptides have been studied. Infrared spectra of high molecular weight poly-L-se&e show that the polypeptide exists in the j? structure in the solid state. ORD$ spectra obtained for films of poly--n-se&e display a negative trough at 233 to 235 rnp and a positive peak centered at 210 to 212 mp, characteristic of the j? form. Low molecular weight poly-L-serine in aqueous solution gives an ORD spectrum characteristic of the random chain. The addition of alcohol and/or cooling of solutions of low molecular weight poly-L-serine results in a transition from a random chain to an ordered structure. ORD evidence suggests the following transition: random -+ c( --+ j3 as a function of alcohol concentration and temperature. A random - /3 transition occurs for higher molecular weight materials, indicating that the transition is dependent on the molecular weight. Block copolymers of the type (rm-s&ne)(L-serine)(nL-serine) have ORD spectra in aqueous solution which resemble that of flhns of high molecular weight poly-L-aerine. These studies implicate the importance of environmental factors in the control of polypeptide chain conformation.
1. Introduction Polypeptide chains fold into relatively few ordered secondary structures: the a helix (Pauling & Corey, 1951a), the /I pleated sheet (Pauling & Corey, 1951b, 1953a) and the poly-L-proline helix (Cowan & McGavin, 1955). The /I conformation has been found in such structural proteins as silk (Huggins, 1943; Pauling & Corey, 19533), stretched horse tail keratin (Fraser & MacRae, 1962) and the egg stalk material of Chrysqva j&a (Parker & Rudall, 1957). Recently, papers describing far ultraviolet ORD studies (Blout & Shechter, 1963; Davidson, Tooney & Fasman, 1966; Sarkar & Doty, 1966; Davidson & Fasman, 1967; Fasman & Potter, 1967; Ikeda & Fasman, 1967) have demonstrated the existence of the ,L?conformation in solution as well as in the solid state. The discovery of a segment of /3 structure in lysozyme (Blake, Mair, North & Phillips, 1967) illustrates the presence of this structure in globular proteins. The theoretical papers of Pysh (1966) and of Rosenheck & Sommer (1967) have provided an initial theoretical framework for the evaluation of the ORD patterns of the /I conformation(s). t Present address: Children’s Cancer Research Found&ion, Inc., 35 Binney Street, Boston, Mass. 02115, U.S.A. $ Abbreviations used: ORD, optical rotatory dispersion; A/I, anhydride to initiator ratio in the polymerization; TFA, tritluoroacetio acid; block copolymer, (m-serine), (L-serine), (DLserine),. 24
355
356
N. M. TOONEY
AND
G. D. FASMAN
Previous X-ray and infrared data had suggested that poly-L-serine derivatives could exist in a ,8 conformation in the solid state (Johnson, 1955; Fasman & Blout, 1960; Bradbury, Elliott & Hanby, 1962; Bohak & Katchalski, 1963; Yahara & Imahori, 1963; Yahara, Imahori, Iitaka & Tsuboi, 1963; Imahori & Yahara, 1964). We present evidence to show that low molecular weight polypeptides of L-serine fold into a /l conformation in aqueous solution under appropriate conditions of alcohol concentration and low temperature. Further, our data indicate that the poly-Lserine in a random chain conformation may at first partially assume the a helical conformation before taking up the /3 conformation under the conditions described below. Block copolymers of the type (on-serine), (L-serine), (DL-serine), synthesized by the technique of Gratzer & Doty (1963) in order to solubilize long sequences of L-serine residues, also form ,6 structures in aqueous solution.
2. Experimental Procedure (a) Solvent8 and reagenti Distilled water was prepared methanol were purchased from
using an all-glass system. Spectral Matheson, Coleman and Bell.
grade
2-propanol
and
(b) Polypeptidea The preparation and characterization of serine have been described elsewhere in this study are listed in Table 1.
of samples of poly-L-serine and copolypeptides (Tooney & Fasman, 1968). The polymers used
TABLE I
Polypeptide
Poly-L-serine Poly-L-s&Ill3 Poly-L-serine POly-DL-SEdne
Poly-0-tert.-butyl-L-serine Poly-0.test.-butyl-L-serine Poly-0.tsrt.-butyl-DL-serine
Sample number
NT-2342 NT-2-376-21 GF-14-437 NT-2-3067 NT-2-376-19 NT-2-292-10 NT-2-304
A/It
200 50 15 200 50 200 200
Viscosityt
1.02 II 0.33 11 0.16(( 0.33 1.11 0.16
Molecular weight §
105,000~ 30,000~ 6507 13,000$ 30,000~ 115,000§ 13,000§
Block oopolymerstt (DL-l313&~
(L-80&,
(DL-BB&,
(DL-se& (L-ser)80 (nL-ser)sO (O-tert.-butyl-nL-ser~e)loo (0-tert.-butyl-L-serine),, (O-te&-butyl-DL-se&e),, (nL-ser)lOO (L-ser)dl @L-se&
NT-24067 NT-3-13
13008 6001
NT-2-307-10
0.2
1700
NT-2-307-7
0.2
170011
t A/I = anhydride/iuitiator ratio in polymerization, sodium methoxide initiation. $ Specific viscosity of the 0-ted.-butyl polymer measured et 0.2% concentration acetic acid. 5 Molecular weight based on the date of Mitchell, Woodward & Doty (1957). 11Measured on unblocked O-tert.-butyl derivatives. 7 Molecular weight estimated by end-group titration (Rosen, 1957). tt n-Hexylamine initiation.
in dichloro-
CONFORMATION
OF SERINE
357
POLYPEPTIDES
(c) Polarizer A silver chloride polarizer was constructed according to the design of Makas & Shurcliff (1955), modified by Bird t Shurcliff (1959). We thank Dr Makas for advice and assistance in constructing it. Silver chloride sheets of 0.02 in. thickness were purchased from the Harshaw Chemical Company. (d) Infrared measurements Polarized and unpolarized spectra were obtained with a Perkin Elmer model 21 infrared spectrophotometer. To produce polarized spectra, the sample film mounted on a silver chloride plate was placed in the optical path ahead of the polarizer. A variable beam attenuator (Connecticut Instruments) was placed in the reference beam to compensate for the loss in transmittance due to the silver chloride plates in the polarizer. Polypeptide samples were dissolved in TFA or distilled water. A drop or two of solution was placed on a silver chloride plate and the sample was stroked unidirectionally to prepare oriented films. (e) Optical rotatory dispersion measurementi Two instruments were used for optical rotatory dispersion measurements. The BendixEriccson Polarimatic 62 automatic recording spectropolarimeter (manufactured by Bendix-Ericoson U.K. Ltd. and distributed by the Bendix Corp., Cincinnati Division, Cincinnati 8, Ohio) was purged with nitrogen for measurements made at wavelengths below 220 mp. The sample compartment of the Cary 60 spectropolarimeter was purged for measurements below 220 mp. Both instruments were calibrated with solutions of sucrose (National Bureau of Standards lot no. 6340) in glass-distilled water. Far ultraviolet silica cells of 0.1, 05, 1.0, 5.0 and 10.0 mm path lengths were purchased from the Optical Cell Company of Brentwood, Md. Sparingly soluble polymer samples were homogenized in a Ten Broeck tissue grinder. All solutions or suspensions were filtered through a sintered glass funnel or through Teflon or cellulose acetate Millipore filter pads to avoid optical artifacts due to light scattering. The concentration of polypeptide in solution was determined by Kjeldhal digestion followed by a Nessler determination of ammonium sulfate according to the method of Lang (1958). Except where otherwise noted, the data are reported as (m’), the reduced mean residue rotation (Fasman, 1963). (f) Index of refraction Values of refractive indices needed to calculate (m’) at various wavelengths were obtained from the Sellmeier approximation (Fasman, 1963): (n2 - 1) = As/(hs - Xi) where n is the index of refraction, I\ is the wavelength of interest and A and )\s are constants to be determined. To evaluate the constants, the refractive indices of the following solvent systems were measured at 589, 546 and 436 rnp using a Bausch & Lomb refractometer with sodium and mercury light sources.
50% 75 y0 50% 75% 50% 75%
Solvent system 2-propanol/50°h water (v/v) 2-propano1/25 ye water (v/v) methanol/60°h water (v/v) methanol/25% water (v/v) 3-butanol/bO% water (v/v) 3-butano1/25% water (v/v)
A 0.8396 0.8616 0.7780 0.7770 0.8603 0.8616
ho(w) 98x5 99.4 97.0 96.2 99.2 99.4
3. Results (a) Infrared
measurements
The polarized infrared spectrum of poly-0-tert.-butyl-L-serine (A/I = 200) was obtained from an oriented film cast from TFA. The principal amide I band, found at 1633 cm-l, showed perpendicular dichroism. The amide II band, located at 1504
358
N. M. TOONEY
AND
G. D. FASMAN
cm-l, showed parallel dichroism. The presence of a small shoulder at 1695 cm-l may indicate that the polypeptide chain folds into an antiparallel p conformation (Miyazawa, 1960; Miyazawa & Blout, 1961). Table 2 presents a summary of the principal band locations and the dichroic properties of the 0-tert.-butyl-L-serine polymers. TABLE 2 Infrared spectral properties of serine polypeptides Amide I (cm-l)
Amide II (cm-l)
Poly-0-tert.-butyl-L-serine A/I = 200 NT-2-291-10 cast from TFA
1633 I 1695 shoulder
150411
Poly-0-tert.-butyl-DL-serine A/I = 200 NT-2-304 cast from TFA
1642 1695 shoulder
1512
Block copolymer (0-tert.-butylDL-serine)(O-te7t.-butyl-Lserine)(O-Wt.-butyl-DL-serine) A/I = 100 (100 : 41 : 88) NT-2-307-10 cast from TFA
1639 1695 shoulder
1812
Poly-L-serine A/I = 200 m-2-374-27 cast from TFA
1626 1695 shoulder
151811
Poly-DL-serine A/I = 200 NT-2-306-7 cast from water
1661 1709 shoulder
1636
Block copolymer (DL-serine) (L-serine)(DL-serine) A/I = 36 (35:30:35) NT-2-40&7 cast from 60% aqueous methanol
1626 aud 1653
1522
I Perpendicular dichroism; II parallel dichroism
Infrared measurements were also carried out on poly-L-serine, poly-DL-serine and a block copolymer of L- and DL-serine. Efforts to prepare oriented films of poly-Lserine cast from TFA were only partly successful. The amide I band of poly-L-serine (A/I = 200) located at 1626 cm-’ showed little or no dichroism; however, the amide II band found at 1518 cm-’ did show parallel dichroism, indicating the /3 structure. A small shoulder at 1695 cm-l was observed. The spectrum of poly-DL-serine cast from 50% aqueous methanol (v/v) was found to be non-dichroic. The amide I and II bands for this polymer were found at 1661 cm-l and 1536 cm-l, respectively. A small shoulder was noted at 1709 cm- I. The infrared spectrum of the block copolymer (DL-serine),, (L-serine),, (DL-Serine)S5 cast from water showed two poorly resolved bands in the amide I region, one centered at 1653 cm-’ and one centered at 1626 cm-‘. The broad amide II band was found at 1522 cm-l. Table 2 summarizes the locations of the amide I and amide II bands for these polyamino acids.
CONFORMATION
OF
SERINE
POLYPEPTIDES
350
(b) Optical rotatory dispersion measurements of $1~ Optical rotatory dispersion measurements of lilms of 0-tert.-butyl-serine polypeptides and serine polypeptides were carried out on thin-film samples. Since it was not possible to measure accurately the polymer concentration of the films, the values of optical rotation have been expressed in terms of cr,,,. The Verdet ratio correction term was applied to spectra obtained on the Bendix-Ericcson instrument. The optical rotatory dispersion spectra of two samples of poly-O-tert.-butyl-Lserine cast from TFA are shown in Figure 1. The spectrum measured on the Bendix-
1
I
I
!
1
I
I
I
I
190 200 210 220 230 240 250 260 270 il (mp) FIG. 1. Optical rotatory dispersion of poly-L-serine and poly-O-tirt.-butyl-L-serine films. Curve A, () poly-L-serine (A/I = 50) measured on 8 Bendix-Ericcson spectropolarimeter; curve B, (-..--) poly-O-tert.-butyl-L-serine (A/I = 50) measured on a BendixEriccson spectropolerimeter; curve C, (-----) poly-O-tert.-butyl-L-serine (A/I = 200) meseured on a Gary 60 spectropolarimeter. Polymer samples were cast on quartz discs from TFA solutions at 22°C.
Ericcson spectropolarimeter had a positive peak at 212 rnp; the spectrum measured on the Cary 60 spectropolarimeter had a positive peak centered at 208 rnp. Since two different polymer preparations were used, the differences observed cannot necessarily be ascribed to instrumental variability. The optical rotatory dispersion spectrum of a film of poly-L-serine is given in Figure 1. The spectrum is characterized by a negative trough near 233 rnp and a positive peak at 210 mp. The spectrum is qualitatively very similar to the spectra obtained for poly-0-tert.-butyl-L-serine. Films of two block copolymers containing both L- and DL-serine were cast from water and examined on the Bendix-Ericcson instrument. Both polymers had spectra quite similar to the spectra obtained for poly-L-serine. These ORD spectra are in agreement with those previously published for the /3 structure (Davidson et al., 1966; Fasman & Potter, 1967).
360
N.
M. TOONEY
AND
G. D. FASMAN
(c) Optical rotutory dispersion meamrementd in solution Polymers of L-se&e were found to be sparingly soluble in water. Samples of poly-L-se&e of varying chain length (A/I = 15 and 50) were measured in 100% aqueous solution. In ah ca8es the ORD spectra were negative over the wavelength region 200 to 300 rnp when measured at 22°C. The presence of a small trough cantered near 230 to 235 rnp wa8 observed in ah cases. Thus, the spectra were qualitatively similar to the spectra reported for the random chain form of poly-Lglutamic acid (Sage BEFasman, 1966) and poly-L-lysine (Greenfield, Davidson & Fasman, 1967). The concentration8 used were in the range O-01 to 0.02%. The spectrum of poly-L-serine (A/I = 15) in water is ahown in Figure 2 (curve A).
FIQ. 2. Optical rotatory dispersion of poly-L-serine (A/I = 15) aa a function of alcohol concentration and temperature. Curve A, (-----) 0.024% in water, S-mm cell, 22.5’C; curve B, (-.-a--) 0.024% in $-propan01 : water 1 : 1 (v/v), 5-mm cell, 22.5’C; curve C, (. . . . . . ) solution used for curve B kept at 6°C for 18hr and measured in a l-mm cell; curve D, (--..-.*--) 0.027% in 2-propanol: water, ) 0.02% in 2-propanol : water, 75 : 25 75 : 25 (v/v), l-mm cell, 22+5”C; curve E, (---(v/v), l-mm cell, -20°C for 18 hr measured at 6°C. [m’]lsa f 2200, [m’],,, -f 300. All samples were measured on a Cary 60 spectropolarimeter.
The addition of 2-propanol to aqueous solution8 of poly-n-serine resulted in pronxmced changes in the ORD spectrum. The ORD spectrum of a 0.024% solution of poly-L-serine (A/I = 15) in 50% 2-propanol/50°/o distilled water (v/v) solution is represented by curve B in Figure 2. The small peak at 220 rnp which occurs in the negative region8 of the spectrum for a lOOo/oaqueous solution shifts to 216 rnp and becomes less negative in 60% 2-propanol/50% aqueous solution (v/v). When this solution was kept at 5°C for 18 hours and then measured again at 22°C a different ORD curve wa8 observed (Fig. 2, curve C). The peak in the region of negative rotation had shifted to 214 mp and became less negative. A cro88-over point wa8 located at 204 rnp and the optical rotation was found to be positive in the 200 to 206 rnp region. An increase in the relative conoentration of isopropanol to 75% by volume resulted in a further shift of the ORD pattern to more positive value8 at wavelengths lower
CONFORMATION
OF
SERINE
POLYPEPTIDES
361
than 220 rnp (Fig. 2, curve D). When this solution, in 75% 2-propano1/25°h water (v/v), was kept at -20°C for 18 hours, a further shift in the ORD spectrum was noted (Fig. 2, curve E) and a peak was found at 196 mp. These experiments suggested that two factors influenced the ORD spectrum of poly-n-serine: temperature and alcohol concentration. These factors were examined independently as well as in combination. The effect of increasing alcohol concentrations upon the ORD spectrum of poly-L-serine (A/I = 15) solutions at 22°C is seen in Figure 2, curves A, B and D. The effect of cooling to -20°C and changing polymer concentration in aqueous media is seen in Figure 3. The ORD spectrum of poly-L-serine (A/I = 15, 0.11%) in water obtained for a solution kept at -20°C for 18 hours is seen in Figure 3, curve A. When a portion of this solution (0.11%) was diluted to O-026% in distilled water and maintained at -20°C for 18 hours, the optical rotatory dispersion, measured at 6”C, yielded curve B of Figure 3. The
I
I
I
195 200
I
I
210 220
1
I
230 240
250
4 (mp)
FIG. 3. Optical rotatory
dispersion
of poly-~-se&e
(A/I = 15) iu water upon cooling at various concentrations. Curve A, (--A--A-) 0.11% solution iu water; ourveB, (-O-O-) 0.020% solution in water; curve C, (--o-n--) O+O13o/osolution iu water. All samples were measured on a Cary 60 spectropolarimeter in a l-mm cell. All samples were stored at -20% for 18 hr prior to measurement at 6°C.
spectrum was characterized by a negative trough at 233 rnp, a cross-over point at 220 rnp, a pronounced shoulder at 210 rnp, and a large positive peak near 194 rnp. This curve is qualitatively similar to that obtained for the sample in 75% 2-propanol/ 25% water (v/v) (Fig. 2, curve E). The sample used to obtain curve B of Figure 3 was then diluted to a concentration of O*013°/0 in distilled water, placed at -20°C overnight and measured at 6°C in a l-mm jacketed cell (Fig. 3, curve C). Although the 210 rnp shoulder remained unchanged, the magnitude of the positive peak at 194 rnp was markedly decreased, indioating a concentration effect.
362
N.
M. TOONEY
AND
G. D. FASMAN
The ORD spectrum of poly-L-serine (A/I = 15) obtained from measurements of cooled aqueous 2-propanol solutions could be altered by an increase of temperature (Fig. 4, curves 1, 2, 3). Curve 1 of Figure 4 shows the ORD spectrum obtained for a O-O25o/osolution of poly-L-se&e which was kept at - 20°C overnight and measured at 22°C. The trough at 235 m/l had a value of (m’)335 of -1360” and the peak at 211 rnp had a value of (m’),,, of +3280. Curve 2 shows the ORD spectrum of the same solution heated at 35°C for 30 minutes. Curve 3 was obtained by heating the solution at 60°C for five minutes. Thus, heating produces a small decrease in the
I
I
I
I
I
I
1
1
190 200 210 220 230 240 250 260 270
FIQ. 4. Optical rotatory dispersion of poly-L-serine (A/I = 15) in 2-propanol : water, 75: 25 (v/v) measured w a function of temperature. curve 1, (. . . . . . ) 0.026% is kept at -20°C for 18 hr, measured at 22°C; curve 2, (-----) solution from curve 1 heated at 35% for 30 min, measured at 35°C; curve 3, (--a -* -) solution from curve 2 heated at 60°C for 6 min, measured at 60°C; curve 4, (--) unheated portion of sample used for curve 1 cooled at -20°C for several days, measured at 22°C. [w&,,,, & 500,
[7G?.asf 200. Measurements
made on a Cary 60 spectropolarimeter
in a l-mm
path length
cell.
magnitude of the 235 rnp trough value, whereas the 210 rnp peak value decreases markedly. A second trough near 204 rnp was observed for curves 1 to 3. Storage of a portion of the unheated solution used to obtain curve 1 at -20°C for several days resulted in an altered ORD curve (Fig. 4, curve 4). The trough became more negative and shifted to 233 mp, while the peak shifted to 209 rnp with a value of (m’) of about 3000”. In contrast to curves 1 to 3, a cross-over point was noted near 198 rnp for curve 4. It was consistently observed that prolonged storage of poly-L-serine solutions at -20°C resulted in ORD spectra similar to that of curve 4, Figure 4. The cross-over point in the region of 198 rnp and the positive peak centered near 210 rnp are similar to the ORD spectra obtained from poly-L-serine in the solid state.
CONFORMATION
OF
SERINE
POLYPEPTIDES
363
ORD studies of poly-L-serine (A/I = 15) were also carried out in aqueous methanol and aqueous tertiary butanol. In all cases, the effects of alcohol concentration and temperature were found to be similar to the effects noted above for aqueous 2-propanol solutions of poly-L-serine. The spectral changes observed with “aging” of solutions at -20°C (Fig. 4, curve 4, for example) were also noted in aqueous methanol and aqueous 3-butanol solutions of poly-L-serine. Measurements were carried out on a sample of higher molecular weight poly-Lscrine (A/I = 50). The optical rotatory dispersion of a 0.01 o/osolution of poly-L-serine (A/I = 50) in water at 22°C is shown in curve 1 of Figure 5. Upon dilution with 2-propanol to yield a O*O075°h solution of the A/I = 50 polypeptide in 50% aqueous 2-propanol, and after storage for 18 hours at -2O”C, the ORD curve 2 in Figure 5 was obtained. The spectra indicate that alcohol and low temperatures favor the formation of a j3 structure by this higher molecular weight material.
190 200 210 220 230 240 2.50 260 270 A(rnp),
Fra. 5. Optical rotatory dispersion of poly-L-serine (A/I = 50). Curve 1, 0.01% solution in water, 22”C, measuredon a Bendix-Ericcson spectropolarimeter; curve 2, 0.0075% solution in 2-propanol : water, 1: 1 (v/v) kept at -20°C for 18hr, measured on a Cery 80 speotropolarimeterat 22°C. Sampleswere measuredin a l-cm cell. (d) Optical rotatory dispersion measurements of block copolymers In Figure 6 is presented the ORD curve obtained for a 0*105% solution of the block copolymer (DL-serine),, (L-serine),, (nL-serine),, (mol. wt approx. 1300) in distilled water. The spectrum exhibits a negative trough at 233 rnp, a cross-over point at 224 rnp and a positive peak near 213 mp. Although the measurements were made under conditions which tax the reliability of the spectropolarimeter (high absorbance, low rotatory strength), the occurrence of positive rotation at wavelengths below 224 rnp is qualitatively correct. This spectrum is similar to those obtained for films of poly-L-serine and to some of the spectra obtained for poly-L-serine in aqueous alcohol solution, indicating the /3 structure. Several optical rotatory dispersion measurements were performed on samples of the block copolymer (nL-serine),, (L-serine),, (DL-serine),, using the Bendix-Ericcson instrument. The rotatory properties of this material were found to be quite similar to that of the block copolymer (35 : 30 : 35) discussed in the preceding paragraph, Measurements were also made on solutions of the (30 : 30 : 30) (mol. wt approx. 600)
364
N. M. TOONEY
AND
G. D. FASMAN
block polymer using a Cary 60 speotropolarimeter. (We thank Professor S. Beychok and Dr D. Urry for several ORD measurements run to corroborate those obtained in our laboratory.) A good agreement between the two sets of measurements was observed for the wavelength region 215 to 260 mp. Extensive studies have not been carried out on the effects of alcohol on the ORD spectra of the block copolymers. It was observed that addition of 2-propanol to an aqueous solution of the (30 : 30 : 30) block copolymer to give a final concentration of 0.107% in 60% 2-propanol/50% water (v/v) increased the peak value of [m’lzIO mu from about 1600” to about 4600” (Fig. 7). No further increase was noted upon cooling the aqueous 2-propanol solution to -20°C for 18 hours. 5
I
4
1
1
1’
1
1
4-
iyo 200 210 220 230 -240 250 260 270 rt tmpL) Fra. 6. Optioal rotatory dispersion of block copolymer (DL-S0rinS)S6 (L-s&m),,, (DL-S0rh3)S5 (mol. wt approx. 1300). The sample was measured at 0.105% concentration in water in a l-mm cell at 24°C on a Bendix-Ericcson spectropolarimeter. s-
4-
3P 52 2* 5-Q s l-
O0,5-
I I I , I 1 lY(3 200 210 220 230 240 250 260
Fro. 7. Optical rotatory dispersion of block copolymer (DL-Serine)30 (I,-serine),,, (DL-serine),, (mol. wt approx. 600). Curve A, O~lSS~o in water at 22°C; curve B, 0.107’$k in 2-propanol : water, 1: 1 (v/v) at 22°C. Samples were measured in a l-mm cell on a Cary 60 spectropolarimeter.
CONFORMATION
OF
SERINE
POLYPEPTIDES
365
4. Discussion The studies reported here were made in an attempt to characterize the optical rotatory dispersion properties of polypeptide chains of L-serine in the /I conformation both in the solid state and in solution. Higher molecular weight poly-L-serine polymers are insoluble (Bohak & Katchalski, 1963) in aqueous media whereas low molecular weight samples are soluble. Initial attempts to circumvent the vexing problem of solubilization of /3 structures led to the synthesis of block copolymers of the type (DL-serine),(L-serine), (DL-serine), where the DL terminal blocks confer solubility. A second approach to the problem utilized the effects of alcohol on low molecular weight L-se&e polymers to promote an ordered conformation. One difficulty encountered in the study of block copolymers which contained DL-amino acid blocks is that the optically inactive material contributes to the absorbance of the solution, although it does not contribute to the optical activity of the solution. The most useful preparations were those in which the ratios of L-serine to DL-serine were in the range of O-3311to l/l. The block nature of these polypeptides has been demonstrated by their solubility properties (Table 4, Tooney & Fasman, 1968), as well as by their infrared spectra, which showed amide I and II band positions characteristic of both L-serine and DL-serine (Table 2). The infrared band locations and the dichroism of the polarized spectra obtained from films of poly-0-tert..butyl-L-serine and poly-L-serine (Table 2) are characteristic of the /3 conformation (Miyazawa, 1960). The presence of a small absorption band at 1695 cm-l may indicate an antiparallel chain arrangement (Miyazawa, 1960; Miyazawa & Blout, 1961). The location of the amide II band of poly-O-tert.-butyl-Lserine at 1504 cm-’ is notably lower than the amide II band assignment of Miyazawa for either the parallel or the antiparallel jl conformations. However, the amide II band of poly-0-acetyl-L-serine has been previously observed at 1514 cm-l (Yahara & Imahori, 1963) and a value of 1503 cm-’ was reported for one p form of poly-Scarboxy oysteine (Elliott, Fraser, MacRae, Stapleton & Suzuki, 1964). The amide I band of poly-L-serine located at 1626 cm-’ is consistent with previous literature values (Fasman $ Blout, 1960; Bohak & Katchalski, 1963). The amide II band for poly-L-serine was found at 1518 cm-l, in contrast with values of 1525 to 1530 cm-l previously cited for this polymer (Blout, de Laze, Bloom & Fasman, 1960; Bohak & Katchalski, 1963). The small amount of dichroism observed for preparations of poly-L-serine may be due to the fact that these materials were not of high molecular weight (Tooney & Fasman, 1968). The infrared spectra of poly-o-tert.-butyl-DLserine and poly-DL-serine revealed that the amide I and II bands were shifted to higher wavenumbers, compared to the corresponding L-polypeptides. The infrared spectra of block copolymers had amide I and II bands located where one might expect a mixture of L and DL residues to absorb. The infrared studies presented above indicated that these preparations of poly-Lserine and poly-0-tert.-butyl-L-serine assume a /l conformation in the solid state. Significantly, ORD measurements carried out on films of the same polypeptide samples gave spectra characterized by a negative trough at 233 rnp and a positive peak located near 210 rnp as reported previously (Davidson et al., 1966; Fasman & Potter, 1967). The shape of these curves is markedly different from the curves attributable to the u helix, the random chain or the collagen-type helix, and, in conjunction with the infrared measurements, is assigned to a /I conformation (Fig. 1).
366
N. M. TOONEY
AND
G. D. FASMAN
Qualitatively, the spectra in Figure 1 are quite similar to those recently observed for a j3 form in solution of high molecular weight poly-L-lysine (Davidson et al., 1966; Davidson & Fasman, 1967; Sarkar & Doty, 1966), for poly&carboxymethyl cysteine (Ikeda & Fasman, 1967) and other &forming polyamino acids in the solid state (Fasman & Potter, 1967). In contrast to the analysis of the ORD spectra of films of poly-L-serine, the interpretation of the spectra of low molecular weight poly-L-serine in solution is more complex. Our observations are summarized below. (1) Low molecular weight poly-L-serine in 100% aqueous solution is primarily in a random chain conformation. (2) A transformation of random chain poly-L-serine into ordered conformations can be accomplished by two procedures: the addition of alcohol to these solutions, or cooling the solutions. These effects are most striking when carried out together. (3) The optical rotatory dispersion evidence suggests that the transition from the random chain conformation to the /I conformation observed for solutions of poly-Lserine may proceed via partial a-helix formation, (4) Higher molecular weight samples of poly-L-serine or of block copolymers attain only random or /I structures. (5) The heating of solutions of poly-L-serine in an ordered conformation results in a decrease of ordered structure, as evidenced by changes in the ORD spectra. The data presented are not sufficiently precise to permit us to make a quantitative assessment of rotatory strength as a function of wavelength, although, clearly, this is of importance. An examination of the rotatory strengths of the various spectra as a function of wavelength indicates that the value of [m’],,,, is -1500 f 250”, regardless of solvent system. Values obtained for peaks and shoulders in the regions of 208 to 212 rnp vary from +1600” to +3000” in water and attain values near +4600” in aqueous alcohol solutions. In circumstances where a positive peak was noted in the wavelength region 194 to 196 rnp, the observed value of [m’] had a value of +15,000” f 2500”. The values of [m’]233ma and [m’]ZIDmfi are no more than half the values which have been obtained for high molecular weight /I poly-L-lysine (Davidson & Fasman, 1967). However, our values were similar to the values found for poly-S-carboxymethyl-L-cysteine (Ikeda & Fasman, 1967). Recent studies of non-helical proteins (Jirgensons, 1967) have indicated that alcohol can promote the formation of a /3 conformation. Similar data have been offered for silk fibroin (Iizuka $ Yang, 1966). It is not surprising, then, to find a similar effect for serine polypeptides. The effect of organic solvents in stabilizing the a-helical conformation is also well-documented (for example, Tanford, Buckley, De & Lively, 1962; Fasman, Lindblow 6 Bodenheimer, 1964; Kientz & Bigelow, 1966; Davidson $ Fasman, 1967). The analysis of the tertiary structure of myoglobin had indicated that serine residues can exist in a helix and that the side-chain hydroxyl groups are capable of interacting with the amide hydrogen bonds of the a-helix (Kendrew, 1962). Courtaulds molecular models of poly-L-serine may readily be formed into an a helix or into parallel or antiparallel ,!l pleated sheets. Although the side-chain oxygen comes close to the peptide group of poly-L-serine in the a-helical conformation by rotation of the serine side-chain, steric considerations do not appear to be limiting. This observation further implicates the role of solvent interactions in stabilizing polypeptide chain conformations. The optical rotatory dispersion measurements of a sample of higher molecular weight poly-n-serine (A/I = 50) in aqueous alcohol solutions indicate that the
CONFORMATION
OF
SERINE
POLYPEPTIDES
367
effects of alcohol and of low temperature produce only a fi conformation (Fig. 5). It would thus appear that the ability of a polypeptide chain to fold into a /I conformation is partly dependent upon molecular weight. The effects of alcohol on the ORD spectrum of serine block copolymers indicate that the /%conformation is promoted by the addition of alcohol (Fig. 7). None of the block copolymers studied gave ORD spectra characteristic of the u helix. The addition of alcohol to solutions of block copolymers had a relatively small effect on the rotatory strength of the 233 rnp trough, but a dramatic effect on the magnitude of the optical rotation at 210 rnp, suggesting that the polypeptide chain is more completely in a /I conformation in alcoholic solution. Higher molecular weight block copolymers also fold more completely into /? structures than do lower molecular weight samples. We offer the following qualitative explanation of our observations. The optical rotatory dispersion and infrared spectra of films of poly-L-serine show that this polypeptide chain takes up a /3 conformation in the absence of solvent. The block copolymers studied are able to assume a ,9 conformation in lOOo/oaqueous solution. Possibly the DL residues serve to solubilize the polypeptide chain, allowing intrachain folding, or the DL residues may shield the middle block of L residues from solvent interactions. Low molecular weight poly-L-serine in lOOo/o aqueous solution probably exists in the random chain conformation because water is a very effective solvating agent for the amide groups, the side-chain hydroxyl groups and the terminal amino and carboxyl groups. The longer chain (A/I = 50) is less likely to be affected by solvation of the terminal groups. Further, the length of the flexible polypeptide chain may give rise to self-shielding from the solvent, thus allowing the stabilization of the molecule by intramolecular folding into a fl conformation. The concentration of block copolymer solutions or of the A/I = 50 poly-n-serine solutions could not be varied over a wide enough range to infer whether the polypeptide chains form interor intramolecular (cross p) structures. The addition of alcohol to aqueous solutions of low molecular weight poly-L-serine results in a decrease in the solvating capacity of the bulk solvent. Also, decreasing the temperature of the solutions may decrease the effective solvating capacity of water because water molecules tend to selfaggregate into “ice-like” clusters at low temperature. Hence, under these conditions, the poly-L-serine chains are able to form into ordered secondary structures, Our data indicate that the low molecular weight poly-L-serine in cold, aqueous alcoholic solution eventually forms a /3 structure, probably the thermodynamically more stable form. Since the polypeptide chains are relatively short, the /3 structure formed may well be intermolecular. Very preliminary studies suggest that the ORD spectra show concentration dependence, as would be expected for intermolecular aggregation. A significant fraction of the molecules appear temporarily to form cchelices by ORD criteria. However, studies with molecular models indicate that it is quite probable that the side-chain hydroxyl groups destabilize the intramolecular hydrogen bonds of the u helix. Since this kind of interference is minimized in the /3 conformations, the molecules all eventually form /3 structures. The significance of the proximity of the side-chain hydroxyl group to the peptide backbone is further emphasized by the fact that the next higher homolog of serine, homoserine, readily folds into an z-helix (Fasman & Blout, unpublished data). Recently, it has been reported that valine polypeptides are also capable of existing in the a helical conformation in methanolic solution (Ooi, Scott, Vanderkooi, Epand
368
N. M. TOONEY
AND
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& Scheraga, 1966), although previous solid-state studies had indicated a /3 structure for poly-n-valine (Blout et al., 1960; Bloom, Fasman, de Laze & Blout, 1962). A tetrapeptide containing a L-Val-L-Val sequence has been suggested possibly to assume the /? conformation in methanol (Shields & McDowell, 1967). These studies further emphasize the important role that solvent takes in determining conformation. Although it has been implied that antiparallel and parallel p conformations have different optical properties in the far ultraviolet region (Pysh, 1966; Rosenheck & Sommer, 1967), a detailed prediction of similarities or differences between the ORD patterns of parallel and antiparallel p structures has not yet been made. Thus a final explanation of our experiments will be contingent upon future theoretical developments. This work was supported in part by grants from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U.S. Public Health Service (AM 0582), the National Science Foundation (GB 5576) and the U.S. Army Medical Research and Development Command, Department of the Army under Research Contract DA 49193.MD-2933. The paper is Publication no. 690 presented in part at the VII International Biochemistry Congress in Tokyo, August 1967. Also this work was done with the support to one of us (N. M. T.) of a National Institutes of Health training grant (26212) and a Public Health Service fellowship (5-Fl-GM 22,161). It was submitted in partial fulfillment of the requirements for the Ph.D. degree at Brandeis University.
REFERENCES Bird, G. R. t Shurcliff, W. A. (1959). J. Opt. Sot. Amer. 49, 235. Blake, C. C. F., Mair, G. A., North,
A. C. T. t Phillips,
D. C. (1967). Proc. Roy. Sot. B,
167, 356.
Bloom, S. M., Fasman, G. D., de Laze, C. & Blout, E. R. (1962). J. Amer. Chem. Sot. 84, 458. Blout, E. R., de Laze, C., Bloom, S. M. $ Fasman, G. D. (1960). J. Amer. Chem. Sot. 82,
3787. Blout, E. R. & Shechter, E. (1963). Btipolymers,
1, 568. Bohak, Z. t Katchalski, E. (1963). Biochem. 2, 228. Bradbury, E. M., Elliott, A. BEHanby, W. E. (1962). J. Mol. Biol. 5, 487. Cowan, P. M. & McGavin, S. (1955). Nacture, 176, 501. Davidson, B. & Fasman, G. D. (1967). B&hem. 6, 1616. Davidson, B., Tooney, N. t Fasman, G. D. (1966). B&hem. Biophys. Rec. Ccwnm. 23,166. Elliott, A., Fraser, R. D. B., MaoRae, T. P., Stapleton, I. W. & Suzuki, E. (1964). J. Mol. BioZ. 9, 10. Fasman, G. D. (1963). MetWe in Enzymology, 6, 928. Fasman, G. D. & Blout, E. R. (1960). J. Amer. Chem. Sot. 82, 2262. Fasman, G. D., Lindblow, C. & Bodenheimer, E. (1964). Biochem, 3, 155. Fasman, G. D. & Potter, J. (1967). B&hem. Biophys. Res. Comm. 27, 209. Fraser, R. D. B. & MacRae, T. P. (1962). J. Mol. BioZ. 5,467. Gratzer, W. B. t Doty, P. (1963). J. Amer. Chsm. Sot. 85, 1193. Greenfield, N., Davidson, B. & Fasman, G. D. (1967). Biochem. 6, 1630. Huggins, M. L. (1943). Chem. Rewe. 32, 195. Iizuka, E. & Yang, J. T. (1966). Proc. Nat. Acad. Sci., Vaah. 55, 1175. Ikeda, S. t Fasman, G. D. (1967). J. Mol. Bid. 30, 491. Imahori, K. & Yshara, I. (1964). Biopolymers Symp. 1, 421. Jirgensons, B. (1967). J. BioZ. Chem. 242, 912. Johnson, C. (1966). Ph.D. thesis, Massachusetts Institute of Technology. Kendrew, J. C. (1962). Brookhaven Symposium, 15, 216. Kientz, M. L. & Bigelow, C. C. (1966). B&hem. 5, 3494. Lang, C. A. (1968). And. Chem. 80, 1692.
CONFORMATION
OF
SERINE
POLYPEPTIDES
369
Makas, A. S. & Shurcliff, W. A. (1955). J. Opt. Sot. Amer. 45, 998. Mitchell, J. C., Woodward, R. B. t Doty, P. (1957). J. Amer. Chem. Sot. 79, 3955. Miyazawa, T. (1960). J. Chem. Phys. 32, 1647. Miyazawa, T. & Blout, E. R. (1961). J. Amer. Chem. Sot. 83, 712. Ooi, T., Scott, R., Vanderkooi, G., Epand, R. F. & Scheraga, H. A. (1966). J. Amer. Chem. Sot. 88, 5680. Parker, K. D. & Rudall, K. M. (1957). Nature, 179, 905. Pauling, L. & Corey, R. B. (1951a). Proc. Nat. Acad. Sci., Wash. 37, 235. Pauling, L. & Corey, R. B. (1951b). Proc. Nut. Accd Sci., Wacrh. 37, 251. Pauling, L. & Corey, R. B. (1953a). Proc. Nat. Acad. Sci., Wash. 39, 253. Pauling, L. & Corey, R. B. (1953b). Proc. Roy. Sot. B, 141, 21. Pysh, E. S. (1966). Proc. Nat. Acad. Sci., Wash. 56, 825. Rosen, H. (1957). Arch. B&hem. Biophys. 67, 10. Rosonheck, K. & Sommer, B. (1967). J. Chem. Phy8. 46, 532. Sage, H. J. & Fasman, G. D. (1966). Biochem. 5, 286. Sarkar, P. & Doty, P. (1966). Proc. Nat. Acad. Sci., Wash. 55, 981. Shields, J. E. & McDowell, S. T. (1967). J. Amer. Chem. Sot. 89, 2499. Tanford, C., Buckley, C. E., De, P. K. & Lively, E. P. (1962). J. Bid. Chem. 237, 1168. Tooney, N. M. & Fasman, G. D. (1968). Riopo~ymers, 6, 81. Yahara, I. & Imahori, K. (1963). J. Amer. Chem. Sot. 85, 230. Yahara, I., Imahori, K., Iitaka, Y. & Tsuboi, M. (1963). J. Polymer Sci. Bl, 47.
Conformation of Serine Polypeptides Optical Rotatory Dispersion and Infrared Studies NANCY M. TOONEYt
AND GERALD D. FASMAN
Graduate Department of Biochemistry, Brandeis University Waltham, .itfa88achu8ett8 02154, U.S.A. (Received 12 February 1968, and in revised form 22 May 1968) The infrared and optical rotatory dispersion spectra of L-serine polypeptides have been studied. Infrared spectra of high molecular weight poly-L-se&e show that the polypeptide exists in the j? structure in the solid state. ORD$ spectra obtained for films of poly--n-se&e display a negative trough at 233 to 235 rnp and a positive peak centered at 210 to 212 mp, characteristic of the j? form. Low molecular weight poly-L-serine in aqueous solution gives an ORD spectrum characteristic of the random chain. The addition of alcohol and/or cooling of solutions of low molecular weight poly-L-serine results in a transition from a random chain to an ordered structure. ORD evidence suggests the following transition: random -+ c( --+ j3 as a function of alcohol concentration and temperature. A random - /3 transition occurs for higher molecular weight materials, indicating that the transition is dependent on the molecular weight. Block copolymers of the type (rm-s&ne)(L-serine)(nL-serine) have ORD spectra in aqueous solution which resemble that of flhns of high molecular weight poly-L-aerine. These studies implicate the importance of environmental factors in the control of polypeptide chain conformation.
1. Introduction Polypeptide chains fold into relatively few ordered secondary structures: the a helix (Pauling & Corey, 1951a), the /I pleated sheet (Pauling & Corey, 1951b, 1953a) and the poly-L-proline helix (Cowan & McGavin, 1955). The /I conformation has been found in such structural proteins as silk (Huggins, 1943; Pauling & Corey, 19533), stretched horse tail keratin (Fraser & MacRae, 1962) and the egg stalk material of Chrysqva j&a (Parker & Rudall, 1957). Recently, papers describing far ultraviolet ORD studies (Blout & Shechter, 1963; Davidson, Tooney & Fasman, 1966; Sarkar & Doty, 1966; Davidson & Fasman, 1967; Fasman & Potter, 1967; Ikeda & Fasman, 1967) have demonstrated the existence of the ,L?conformation in solution as well as in the solid state. The discovery of a segment of /3 structure in lysozyme (Blake, Mair, North & Phillips, 1967) illustrates the presence of this structure in globular proteins. The theoretical papers of Pysh (1966) and of Rosenheck & Sommer (1967) have provided an initial theoretical framework for the evaluation of the ORD patterns of the /I conformation(s). t Present address: Children’s Cancer Research Found&ion, Inc., 35 Binney Street, Boston, Mass. 02115, U.S.A. $ Abbreviations used: ORD, optical rotatory dispersion; A/I, anhydride to initiator ratio in the polymerization; TFA, tritluoroacetio acid; block copolymer, (m-serine), (L-serine), (DLserine),. 24
355
356
N. M. TOONEY
AND
G. D. FASMAN
Previous X-ray and infrared data had suggested that poly-L-serine derivatives could exist in a ,8 conformation in the solid state (Johnson, 1955; Fasman & Blout, 1960; Bradbury, Elliott & Hanby, 1962; Bohak & Katchalski, 1963; Yahara & Imahori, 1963; Yahara, Imahori, Iitaka & Tsuboi, 1963; Imahori & Yahara, 1964). We present evidence to show that low molecular weight polypeptides of L-serine fold into a /l conformation in aqueous solution under appropriate conditions of alcohol concentration and low temperature. Further, our data indicate that the poly-Lserine in a random chain conformation may at first partially assume the a helical conformation before taking up the /3 conformation under the conditions described below. Block copolymers of the type (on-serine), (L-serine), (DL-serine), synthesized by the technique of Gratzer & Doty (1963) in order to solubilize long sequences of L-serine residues, also form ,6 structures in aqueous solution.
2. Experimental Procedure (a) Solvent8 and reagenti Distilled water was prepared methanol were purchased from
using an all-glass system. Spectral Matheson, Coleman and Bell.
grade
2-propanol
and
(b) Polypeptidea The preparation and characterization of serine have been described elsewhere in this study are listed in Table 1.
of samples of poly-L-serine and copolypeptides (Tooney & Fasman, 1968). The polymers used
TABLE I
Polypeptide
Poly-L-serine Poly-L-s&Ill3 Poly-L-serine POly-DL-SEdne
Poly-0-tert.-butyl-L-serine Poly-0.test.-butyl-L-serine Poly-0.tsrt.-butyl-DL-serine
Sample number
NT-2342 NT-2-376-21 GF-14-437 NT-2-3067 NT-2-376-19 NT-2-292-10 NT-2-304
A/It
200 50 15 200 50 200 200
Viscosityt
1.02 II 0.33 11 0.16(( 0.33 1.11 0.16
Molecular weight §
105,000~ 30,000~ 6507 13,000$ 30,000~ 115,000§ 13,000§
Block oopolymerstt (DL-l313&~
(L-80&,
(DL-BB&,
(DL-se& (L-ser)80 (nL-ser)sO (O-tert.-butyl-nL-ser~e)loo (0-tert.-butyl-L-serine),, (O-te&-butyl-DL-se&e),, (nL-ser)lOO (L-ser)dl @L-se&
NT-24067 NT-3-13
13008 6001
NT-2-307-10
0.2
1700
NT-2-307-7
0.2
170011
t A/I = anhydride/iuitiator ratio in polymerization, sodium methoxide initiation. $ Specific viscosity of the 0-ted.-butyl polymer measured et 0.2% concentration acetic acid. 5 Molecular weight based on the date of Mitchell, Woodward & Doty (1957). 11Measured on unblocked O-tert.-butyl derivatives. 7 Molecular weight estimated by end-group titration (Rosen, 1957). tt n-Hexylamine initiation.
in dichloro-
CONFORMATION
OF SERINE
357
POLYPEPTIDES
(c) Polarizer A silver chloride polarizer was constructed according to the design of Makas & Shurcliff (1955), modified by Bird t Shurcliff (1959). We thank Dr Makas for advice and assistance in constructing it. Silver chloride sheets of 0.02 in. thickness were purchased from the Harshaw Chemical Company. (d) Infrared measurements Polarized and unpolarized spectra were obtained with a Perkin Elmer model 21 infrared spectrophotometer. To produce polarized spectra, the sample film mounted on a silver chloride plate was placed in the optical path ahead of the polarizer. A variable beam attenuator (Connecticut Instruments) was placed in the reference beam to compensate for the loss in transmittance due to the silver chloride plates in the polarizer. Polypeptide samples were dissolved in TFA or distilled water. A drop or two of solution was placed on a silver chloride plate and the sample was stroked unidirectionally to prepare oriented films. (e) Optical rotatory dispersion measurementi Two instruments were used for optical rotatory dispersion measurements. The BendixEriccson Polarimatic 62 automatic recording spectropolarimeter (manufactured by Bendix-Ericoson U.K. Ltd. and distributed by the Bendix Corp., Cincinnati Division, Cincinnati 8, Ohio) was purged with nitrogen for measurements made at wavelengths below 220 mp. The sample compartment of the Cary 60 spectropolarimeter was purged for measurements below 220 mp. Both instruments were calibrated with solutions of sucrose (National Bureau of Standards lot no. 6340) in glass-distilled water. Far ultraviolet silica cells of 0.1, 05, 1.0, 5.0 and 10.0 mm path lengths were purchased from the Optical Cell Company of Brentwood, Md. Sparingly soluble polymer samples were homogenized in a Ten Broeck tissue grinder. All solutions or suspensions were filtered through a sintered glass funnel or through Teflon or cellulose acetate Millipore filter pads to avoid optical artifacts due to light scattering. The concentration of polypeptide in solution was determined by Kjeldhal digestion followed by a Nessler determination of ammonium sulfate according to the method of Lang (1958). Except where otherwise noted, the data are reported as (m’), the reduced mean residue rotation (Fasman, 1963). (f) Index of refraction Values of refractive indices needed to calculate (m’) at various wavelengths were obtained from the Sellmeier approximation (Fasman, 1963): (n2 - 1) = As/(hs - Xi) where n is the index of refraction, I\ is the wavelength of interest and A and )\s are constants to be determined. To evaluate the constants, the refractive indices of the following solvent systems were measured at 589, 546 and 436 rnp using a Bausch & Lomb refractometer with sodium and mercury light sources.
50% 75 y0 50% 75% 50% 75%
Solvent system 2-propanol/50°h water (v/v) 2-propano1/25 ye water (v/v) methanol/60°h water (v/v) methanol/25% water (v/v) 3-butanol/bO% water (v/v) 3-butano1/25% water (v/v)
A 0.8396 0.8616 0.7780 0.7770 0.8603 0.8616
ho(w) 98x5 99.4 97.0 96.2 99.2 99.4
3. Results (a) Infrared
measurements
The polarized infrared spectrum of poly-0-tert.-butyl-L-serine (A/I = 200) was obtained from an oriented film cast from TFA. The principal amide I band, found at 1633 cm-l, showed perpendicular dichroism. The amide II band, located at 1504
358
N. M. TOONEY
AND
G. D. FASMAN
cm-l, showed parallel dichroism. The presence of a small shoulder at 1695 cm-l may indicate that the polypeptide chain folds into an antiparallel p conformation (Miyazawa, 1960; Miyazawa & Blout, 1961). Table 2 presents a summary of the principal band locations and the dichroic properties of the 0-tert.-butyl-L-serine polymers. TABLE 2 Infrared spectral properties of serine polypeptides Amide I (cm-l)
Amide II (cm-l)
Poly-0-tert.-butyl-L-serine A/I = 200 NT-2-291-10 cast from TFA
1633 I 1695 shoulder
150411
Poly-0-tert.-butyl-DL-serine A/I = 200 NT-2-304 cast from TFA
1642 1695 shoulder
1512
Block copolymer (0-tert.-butylDL-serine)(O-te7t.-butyl-Lserine)(O-Wt.-butyl-DL-serine) A/I = 100 (100 : 41 : 88) NT-2-307-10 cast from TFA
1639 1695 shoulder
1812
Poly-L-serine A/I = 200 m-2-374-27 cast from TFA
1626 1695 shoulder
151811
Poly-DL-serine A/I = 200 NT-2-306-7 cast from water
1661 1709 shoulder
1636
Block copolymer (DL-serine) (L-serine)(DL-serine) A/I = 36 (35:30:35) NT-2-40&7 cast from 60% aqueous methanol
1626 aud 1653
1522
I Perpendicular dichroism; II parallel dichroism
Infrared measurements were also carried out on poly-L-serine, poly-DL-serine and a block copolymer of L- and DL-serine. Efforts to prepare oriented films of poly-Lserine cast from TFA were only partly successful. The amide I band of poly-L-serine (A/I = 200) located at 1626 cm-’ showed little or no dichroism; however, the amide II band found at 1518 cm-’ did show parallel dichroism, indicating the /3 structure. A small shoulder at 1695 cm-l was observed. The spectrum of poly-DL-serine cast from 50% aqueous methanol (v/v) was found to be non-dichroic. The amide I and II bands for this polymer were found at 1661 cm-l and 1536 cm-l, respectively. A small shoulder was noted at 1709 cm- I. The infrared spectrum of the block copolymer (DL-serine),, (L-serine),, (DL-Serine)S5 cast from water showed two poorly resolved bands in the amide I region, one centered at 1653 cm-’ and one centered at 1626 cm-‘. The broad amide II band was found at 1522 cm-l. Table 2 summarizes the locations of the amide I and amide II bands for these polyamino acids.
CONFORMATION
OF
SERINE
POLYPEPTIDES
350
(b) Optical rotatory dispersion measurements of $1~ Optical rotatory dispersion measurements of lilms of 0-tert.-butyl-serine polypeptides and serine polypeptides were carried out on thin-film samples. Since it was not possible to measure accurately the polymer concentration of the films, the values of optical rotation have been expressed in terms of cr,,,. The Verdet ratio correction term was applied to spectra obtained on the Bendix-Ericcson instrument. The optical rotatory dispersion spectra of two samples of poly-O-tert.-butyl-Lserine cast from TFA are shown in Figure 1. The spectrum measured on the Bendix-
1
I
I
!
1
I
I
I
I
190 200 210 220 230 240 250 260 270 il (mp) FIG. 1. Optical rotatory dispersion of poly-L-serine and poly-O-tirt.-butyl-L-serine films. Curve A, () poly-L-serine (A/I = 50) measured on 8 Bendix-Ericcson spectropolarimeter; curve B, (-..--) poly-O-tert.-butyl-L-serine (A/I = 50) measured on a BendixEriccson spectropolerimeter; curve C, (-----) poly-O-tert.-butyl-L-serine (A/I = 200) meseured on a Gary 60 spectropolarimeter. Polymer samples were cast on quartz discs from TFA solutions at 22°C.
Ericcson spectropolarimeter had a positive peak at 212 rnp; the spectrum measured on the Cary 60 spectropolarimeter had a positive peak centered at 208 rnp. Since two different polymer preparations were used, the differences observed cannot necessarily be ascribed to instrumental variability. The optical rotatory dispersion spectrum of a film of poly-L-serine is given in Figure 1. The spectrum is characterized by a negative trough near 233 rnp and a positive peak at 210 mp. The spectrum is qualitatively very similar to the spectra obtained for poly-0-tert.-butyl-L-serine. Films of two block copolymers containing both L- and DL-serine were cast from water and examined on the Bendix-Ericcson instrument. Both polymers had spectra quite similar to the spectra obtained for poly-L-serine. These ORD spectra are in agreement with those previously published for the /3 structure (Davidson et al., 1966; Fasman & Potter, 1967).
360
N.
M. TOONEY
AND
G. D. FASMAN
(c) Optical rotutory dispersion meamrementd in solution Polymers of L-se&e were found to be sparingly soluble in water. Samples of poly-L-se&e of varying chain length (A/I = 15 and 50) were measured in 100% aqueous solution. In ah ca8es the ORD spectra were negative over the wavelength region 200 to 300 rnp when measured at 22°C. The presence of a small trough cantered near 230 to 235 rnp wa8 observed in ah cases. Thus, the spectra were qualitatively similar to the spectra reported for the random chain form of poly-Lglutamic acid (Sage BEFasman, 1966) and poly-L-lysine (Greenfield, Davidson & Fasman, 1967). The concentration8 used were in the range O-01 to 0.02%. The spectrum of poly-L-serine (A/I = 15) in water is ahown in Figure 2 (curve A).
FIQ. 2. Optical rotatory dispersion of poly-L-serine (A/I = 15) aa a function of alcohol concentration and temperature. Curve A, (-----) 0.024% in water, S-mm cell, 22.5’C; curve B, (-.-a--) 0.024% in $-propan01 : water 1 : 1 (v/v), 5-mm cell, 22.5’C; curve C, (. . . . . . ) solution used for curve B kept at 6°C for 18hr and measured in a l-mm cell; curve D, (--..-.*--) 0.027% in 2-propanol: water, ) 0.02% in 2-propanol : water, 75 : 25 75 : 25 (v/v), l-mm cell, 22+5”C; curve E, (---(v/v), l-mm cell, -20°C for 18 hr measured at 6°C. [m’]lsa f 2200, [m’],,, -f 300. All samples were measured on a Cary 60 spectropolarimeter.
The addition of 2-propanol to aqueous solution8 of poly-n-serine resulted in pronxmced changes in the ORD spectrum. The ORD spectrum of a 0.024% solution of poly-L-serine (A/I = 15) in 50% 2-propanol/50°/o distilled water (v/v) solution is represented by curve B in Figure 2. The small peak at 220 rnp which occurs in the negative region8 of the spectrum for a lOOo/oaqueous solution shifts to 216 rnp and becomes less negative in 60% 2-propanol/50% aqueous solution (v/v). When this solution was kept at 5°C for 18 hours and then measured again at 22°C a different ORD curve wa8 observed (Fig. 2, curve C). The peak in the region of negative rotation had shifted to 214 mp and became less negative. A cro88-over point wa8 located at 204 rnp and the optical rotation was found to be positive in the 200 to 206 rnp region. An increase in the relative conoentration of isopropanol to 75% by volume resulted in a further shift of the ORD pattern to more positive value8 at wavelengths lower
CONFORMATION
OF
SERINE
POLYPEPTIDES
361
than 220 rnp (Fig. 2, curve D). When this solution, in 75% 2-propano1/25°h water (v/v), was kept at -20°C for 18 hours, a further shift in the ORD spectrum was noted (Fig. 2, curve E) and a peak was found at 196 mp. These experiments suggested that two factors influenced the ORD spectrum of poly-n-serine: temperature and alcohol concentration. These factors were examined independently as well as in combination. The effect of increasing alcohol concentrations upon the ORD spectrum of poly-L-serine (A/I = 15) solutions at 22°C is seen in Figure 2, curves A, B and D. The effect of cooling to -20°C and changing polymer concentration in aqueous media is seen in Figure 3. The ORD spectrum of poly-L-serine (A/I = 15, 0.11%) in water obtained for a solution kept at -20°C for 18 hours is seen in Figure 3, curve A. When a portion of this solution (0.11%) was diluted to O-026% in distilled water and maintained at -20°C for 18 hours, the optical rotatory dispersion, measured at 6”C, yielded curve B of Figure 3. The
I
I
I
195 200
I
I
210 220
1
I
230 240
250
4 (mp)
FIG. 3. Optical rotatory
dispersion
of poly-~-se&e
(A/I = 15) iu water upon cooling at various concentrations. Curve A, (--A--A-) 0.11% solution iu water; ourveB, (-O-O-) 0.020% solution in water; curve C, (--o-n--) O+O13o/osolution iu water. All samples were measured on a Cary 60 spectropolarimeter in a l-mm cell. All samples were stored at -20% for 18 hr prior to measurement at 6°C.
spectrum was characterized by a negative trough at 233 rnp, a cross-over point at 220 rnp, a pronounced shoulder at 210 rnp, and a large positive peak near 194 rnp. This curve is qualitatively similar to that obtained for the sample in 75% 2-propanol/ 25% water (v/v) (Fig. 2, curve E). The sample used to obtain curve B of Figure 3 was then diluted to a concentration of O*013°/0 in distilled water, placed at -20°C overnight and measured at 6°C in a l-mm jacketed cell (Fig. 3, curve C). Although the 210 rnp shoulder remained unchanged, the magnitude of the positive peak at 194 rnp was markedly decreased, indioating a concentration effect.
362
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M. TOONEY
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The ORD spectrum of poly-L-serine (A/I = 15) obtained from measurements of cooled aqueous 2-propanol solutions could be altered by an increase of temperature (Fig. 4, curves 1, 2, 3). Curve 1 of Figure 4 shows the ORD spectrum obtained for a O-O25o/osolution of poly-L-se&e which was kept at - 20°C overnight and measured at 22°C. The trough at 235 m/l had a value of (m’)335 of -1360” and the peak at 211 rnp had a value of (m’),,, of +3280. Curve 2 shows the ORD spectrum of the same solution heated at 35°C for 30 minutes. Curve 3 was obtained by heating the solution at 60°C for five minutes. Thus, heating produces a small decrease in the
I
I
I
I
I
I
1
1
190 200 210 220 230 240 250 260 270
FIQ. 4. Optical rotatory dispersion of poly-L-serine (A/I = 15) in 2-propanol : water, 75: 25 (v/v) measured w a function of temperature. curve 1, (. . . . . . ) 0.026% is kept at -20°C for 18 hr, measured at 22°C; curve 2, (-----) solution from curve 1 heated at 35% for 30 min, measured at 35°C; curve 3, (--a -* -) solution from curve 2 heated at 60°C for 6 min, measured at 60°C; curve 4, (--) unheated portion of sample used for curve 1 cooled at -20°C for several days, measured at 22°C. [w&,,,, & 500,
[7G?.asf 200. Measurements
made on a Cary 60 spectropolarimeter
in a l-mm
path length
cell.
magnitude of the 235 rnp trough value, whereas the 210 rnp peak value decreases markedly. A second trough near 204 rnp was observed for curves 1 to 3. Storage of a portion of the unheated solution used to obtain curve 1 at -20°C for several days resulted in an altered ORD curve (Fig. 4, curve 4). The trough became more negative and shifted to 233 mp, while the peak shifted to 209 rnp with a value of (m’) of about 3000”. In contrast to curves 1 to 3, a cross-over point was noted near 198 rnp for curve 4. It was consistently observed that prolonged storage of poly-L-serine solutions at -20°C resulted in ORD spectra similar to that of curve 4, Figure 4. The cross-over point in the region of 198 rnp and the positive peak centered near 210 rnp are similar to the ORD spectra obtained from poly-L-serine in the solid state.
CONFORMATION
OF
SERINE
POLYPEPTIDES
363
ORD studies of poly-L-serine (A/I = 15) were also carried out in aqueous methanol and aqueous tertiary butanol. In all cases, the effects of alcohol concentration and temperature were found to be similar to the effects noted above for aqueous 2-propanol solutions of poly-L-serine. The spectral changes observed with “aging” of solutions at -20°C (Fig. 4, curve 4, for example) were also noted in aqueous methanol and aqueous 3-butanol solutions of poly-L-serine. Measurements were carried out on a sample of higher molecular weight poly-Lscrine (A/I = 50). The optical rotatory dispersion of a 0.01 o/osolution of poly-L-serine (A/I = 50) in water at 22°C is shown in curve 1 of Figure 5. Upon dilution with 2-propanol to yield a O*O075°h solution of the A/I = 50 polypeptide in 50% aqueous 2-propanol, and after storage for 18 hours at -2O”C, the ORD curve 2 in Figure 5 was obtained. The spectra indicate that alcohol and low temperatures favor the formation of a j3 structure by this higher molecular weight material.
190 200 210 220 230 240 2.50 260 270 A(rnp),
Fra. 5. Optical rotatory dispersion of poly-L-serine (A/I = 50). Curve 1, 0.01% solution in water, 22”C, measuredon a Bendix-Ericcson spectropolarimeter; curve 2, 0.0075% solution in 2-propanol : water, 1: 1 (v/v) kept at -20°C for 18hr, measured on a Cery 80 speotropolarimeterat 22°C. Sampleswere measuredin a l-cm cell. (d) Optical rotatory dispersion measurements of block copolymers In Figure 6 is presented the ORD curve obtained for a 0*105% solution of the block copolymer (DL-serine),, (L-serine),, (nL-serine),, (mol. wt approx. 1300) in distilled water. The spectrum exhibits a negative trough at 233 rnp, a cross-over point at 224 rnp and a positive peak near 213 mp. Although the measurements were made under conditions which tax the reliability of the spectropolarimeter (high absorbance, low rotatory strength), the occurrence of positive rotation at wavelengths below 224 rnp is qualitatively correct. This spectrum is similar to those obtained for films of poly-L-serine and to some of the spectra obtained for poly-L-serine in aqueous alcohol solution, indicating the /3 structure. Several optical rotatory dispersion measurements were performed on samples of the block copolymer (nL-serine),, (L-serine),, (DL-serine),, using the Bendix-Ericcson instrument. The rotatory properties of this material were found to be quite similar to that of the block copolymer (35 : 30 : 35) discussed in the preceding paragraph, Measurements were also made on solutions of the (30 : 30 : 30) (mol. wt approx. 600)
364
N. M. TOONEY
AND
G. D. FASMAN
block polymer using a Cary 60 speotropolarimeter. (We thank Professor S. Beychok and Dr D. Urry for several ORD measurements run to corroborate those obtained in our laboratory.) A good agreement between the two sets of measurements was observed for the wavelength region 215 to 260 mp. Extensive studies have not been carried out on the effects of alcohol on the ORD spectra of the block copolymers. It was observed that addition of 2-propanol to an aqueous solution of the (30 : 30 : 30) block copolymer to give a final concentration of 0.107% in 60% 2-propanol/50% water (v/v) increased the peak value of [m’lzIO mu from about 1600” to about 4600” (Fig. 7). No further increase was noted upon cooling the aqueous 2-propanol solution to -20°C for 18 hours. 5
I
4
1
1
1’
1
1
4-
iyo 200 210 220 230 -240 250 260 270 rt tmpL) Fra. 6. Optioal rotatory dispersion of block copolymer (DL-S0rinS)S6 (L-s&m),,, (DL-S0rh3)S5 (mol. wt approx. 1300). The sample was measured at 0.105% concentration in water in a l-mm cell at 24°C on a Bendix-Ericcson spectropolarimeter. s-
4-
3P 52 2* 5-Q s l-
O0,5-
I I I , I 1 lY(3 200 210 220 230 240 250 260
Fro. 7. Optical rotatory dispersion of block copolymer (DL-Serine)30 (I,-serine),,, (DL-serine),, (mol. wt approx. 600). Curve A, O~lSS~o in water at 22°C; curve B, 0.107’$k in 2-propanol : water, 1: 1 (v/v) at 22°C. Samples were measured in a l-mm cell on a Cary 60 spectropolarimeter.
CONFORMATION
OF
SERINE
POLYPEPTIDES
365
4. Discussion The studies reported here were made in an attempt to characterize the optical rotatory dispersion properties of polypeptide chains of L-serine in the /I conformation both in the solid state and in solution. Higher molecular weight poly-L-serine polymers are insoluble (Bohak & Katchalski, 1963) in aqueous media whereas low molecular weight samples are soluble. Initial attempts to circumvent the vexing problem of solubilization of /3 structures led to the synthesis of block copolymers of the type (DL-serine),(L-serine), (DL-serine), where the DL terminal blocks confer solubility. A second approach to the problem utilized the effects of alcohol on low molecular weight L-se&e polymers to promote an ordered conformation. One difficulty encountered in the study of block copolymers which contained DL-amino acid blocks is that the optically inactive material contributes to the absorbance of the solution, although it does not contribute to the optical activity of the solution. The most useful preparations were those in which the ratios of L-serine to DL-serine were in the range of O-3311to l/l. The block nature of these polypeptides has been demonstrated by their solubility properties (Table 4, Tooney & Fasman, 1968), as well as by their infrared spectra, which showed amide I and II band positions characteristic of both L-serine and DL-serine (Table 2). The infrared band locations and the dichroism of the polarized spectra obtained from films of poly-0-tert..butyl-L-serine and poly-L-serine (Table 2) are characteristic of the /3 conformation (Miyazawa, 1960). The presence of a small absorption band at 1695 cm-l may indicate an antiparallel chain arrangement (Miyazawa, 1960; Miyazawa & Blout, 1961). The location of the amide II band of poly-O-tert.-butyl-Lserine at 1504 cm-’ is notably lower than the amide II band assignment of Miyazawa for either the parallel or the antiparallel jl conformations. However, the amide II band of poly-0-acetyl-L-serine has been previously observed at 1514 cm-l (Yahara & Imahori, 1963) and a value of 1503 cm-’ was reported for one p form of poly-Scarboxy oysteine (Elliott, Fraser, MacRae, Stapleton & Suzuki, 1964). The amide I band of poly-L-serine located at 1626 cm-’ is consistent with previous literature values (Fasman $ Blout, 1960; Bohak & Katchalski, 1963). The amide II band for poly-L-serine was found at 1518 cm-l, in contrast with values of 1525 to 1530 cm-l previously cited for this polymer (Blout, de Laze, Bloom & Fasman, 1960; Bohak & Katchalski, 1963). The small amount of dichroism observed for preparations of poly-L-serine may be due to the fact that these materials were not of high molecular weight (Tooney & Fasman, 1968). The infrared spectra of poly-o-tert.-butyl-DLserine and poly-DL-serine revealed that the amide I and II bands were shifted to higher wavenumbers, compared to the corresponding L-polypeptides. The infrared spectra of block copolymers had amide I and II bands located where one might expect a mixture of L and DL residues to absorb. The infrared studies presented above indicated that these preparations of poly-Lserine and poly-0-tert.-butyl-L-serine assume a /l conformation in the solid state. Significantly, ORD measurements carried out on films of the same polypeptide samples gave spectra characterized by a negative trough at 233 rnp and a positive peak located near 210 rnp as reported previously (Davidson et al., 1966; Fasman & Potter, 1967). The shape of these curves is markedly different from the curves attributable to the u helix, the random chain or the collagen-type helix, and, in conjunction with the infrared measurements, is assigned to a /I conformation (Fig. 1).
366
N. M. TOONEY
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G. D. FASMAN
Qualitatively, the spectra in Figure 1 are quite similar to those recently observed for a j3 form in solution of high molecular weight poly-L-lysine (Davidson et al., 1966; Davidson & Fasman, 1967; Sarkar & Doty, 1966), for poly&carboxymethyl cysteine (Ikeda & Fasman, 1967) and other &forming polyamino acids in the solid state (Fasman & Potter, 1967). In contrast to the analysis of the ORD spectra of films of poly-L-serine, the interpretation of the spectra of low molecular weight poly-L-serine in solution is more complex. Our observations are summarized below. (1) Low molecular weight poly-L-serine in 100% aqueous solution is primarily in a random chain conformation. (2) A transformation of random chain poly-L-serine into ordered conformations can be accomplished by two procedures: the addition of alcohol to these solutions, or cooling the solutions. These effects are most striking when carried out together. (3) The optical rotatory dispersion evidence suggests that the transition from the random chain conformation to the /I conformation observed for solutions of poly-Lserine may proceed via partial a-helix formation, (4) Higher molecular weight samples of poly-L-serine or of block copolymers attain only random or /I structures. (5) The heating of solutions of poly-L-serine in an ordered conformation results in a decrease of ordered structure, as evidenced by changes in the ORD spectra. The data presented are not sufficiently precise to permit us to make a quantitative assessment of rotatory strength as a function of wavelength, although, clearly, this is of importance. An examination of the rotatory strengths of the various spectra as a function of wavelength indicates that the value of [m’],,,, is -1500 f 250”, regardless of solvent system. Values obtained for peaks and shoulders in the regions of 208 to 212 rnp vary from +1600” to +3000” in water and attain values near +4600” in aqueous alcohol solutions. In circumstances where a positive peak was noted in the wavelength region 194 to 196 rnp, the observed value of [m’] had a value of +15,000” f 2500”. The values of [m’]233ma and [m’]ZIDmfi are no more than half the values which have been obtained for high molecular weight /I poly-L-lysine (Davidson & Fasman, 1967). However, our values were similar to the values found for poly-S-carboxymethyl-L-cysteine (Ikeda & Fasman, 1967). Recent studies of non-helical proteins (Jirgensons, 1967) have indicated that alcohol can promote the formation of a /3 conformation. Similar data have been offered for silk fibroin (Iizuka $ Yang, 1966). It is not surprising, then, to find a similar effect for serine polypeptides. The effect of organic solvents in stabilizing the a-helical conformation is also well-documented (for example, Tanford, Buckley, De & Lively, 1962; Fasman, Lindblow 6 Bodenheimer, 1964; Kientz & Bigelow, 1966; Davidson $ Fasman, 1967). The analysis of the tertiary structure of myoglobin had indicated that serine residues can exist in a helix and that the side-chain hydroxyl groups are capable of interacting with the amide hydrogen bonds of the a-helix (Kendrew, 1962). Courtaulds molecular models of poly-L-serine may readily be formed into an a helix or into parallel or antiparallel ,!l pleated sheets. Although the side-chain oxygen comes close to the peptide group of poly-L-serine in the a-helical conformation by rotation of the serine side-chain, steric considerations do not appear to be limiting. This observation further implicates the role of solvent interactions in stabilizing polypeptide chain conformations. The optical rotatory dispersion measurements of a sample of higher molecular weight poly-n-serine (A/I = 50) in aqueous alcohol solutions indicate that the
CONFORMATION
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SERINE
POLYPEPTIDES
367
effects of alcohol and of low temperature produce only a fi conformation (Fig. 5). It would thus appear that the ability of a polypeptide chain to fold into a /I conformation is partly dependent upon molecular weight. The effects of alcohol on the ORD spectrum of serine block copolymers indicate that the /%conformation is promoted by the addition of alcohol (Fig. 7). None of the block copolymers studied gave ORD spectra characteristic of the u helix. The addition of alcohol to solutions of block copolymers had a relatively small effect on the rotatory strength of the 233 rnp trough, but a dramatic effect on the magnitude of the optical rotation at 210 rnp, suggesting that the polypeptide chain is more completely in a /I conformation in alcoholic solution. Higher molecular weight block copolymers also fold more completely into /? structures than do lower molecular weight samples. We offer the following qualitative explanation of our observations. The optical rotatory dispersion and infrared spectra of films of poly-L-serine show that this polypeptide chain takes up a /3 conformation in the absence of solvent. The block copolymers studied are able to assume a ,9 conformation in lOOo/oaqueous solution. Possibly the DL residues serve to solubilize the polypeptide chain, allowing intrachain folding, or the DL residues may shield the middle block of L residues from solvent interactions. Low molecular weight poly-L-serine in lOOo/o aqueous solution probably exists in the random chain conformation because water is a very effective solvating agent for the amide groups, the side-chain hydroxyl groups and the terminal amino and carboxyl groups. The longer chain (A/I = 50) is less likely to be affected by solvation of the terminal groups. Further, the length of the flexible polypeptide chain may give rise to self-shielding from the solvent, thus allowing the stabilization of the molecule by intramolecular folding into a fl conformation. The concentration of block copolymer solutions or of the A/I = 50 poly-n-serine solutions could not be varied over a wide enough range to infer whether the polypeptide chains form interor intramolecular (cross p) structures. The addition of alcohol to aqueous solutions of low molecular weight poly-L-serine results in a decrease in the solvating capacity of the bulk solvent. Also, decreasing the temperature of the solutions may decrease the effective solvating capacity of water because water molecules tend to selfaggregate into “ice-like” clusters at low temperature. Hence, under these conditions, the poly-L-serine chains are able to form into ordered secondary structures, Our data indicate that the low molecular weight poly-L-serine in cold, aqueous alcoholic solution eventually forms a /3 structure, probably the thermodynamically more stable form. Since the polypeptide chains are relatively short, the /3 structure formed may well be intermolecular. Very preliminary studies suggest that the ORD spectra show concentration dependence, as would be expected for intermolecular aggregation. A significant fraction of the molecules appear temporarily to form cchelices by ORD criteria. However, studies with molecular models indicate that it is quite probable that the side-chain hydroxyl groups destabilize the intramolecular hydrogen bonds of the u helix. Since this kind of interference is minimized in the /3 conformations, the molecules all eventually form /3 structures. The significance of the proximity of the side-chain hydroxyl group to the peptide backbone is further emphasized by the fact that the next higher homolog of serine, homoserine, readily folds into an z-helix (Fasman & Blout, unpublished data). Recently, it has been reported that valine polypeptides are also capable of existing in the a helical conformation in methanolic solution (Ooi, Scott, Vanderkooi, Epand
368
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& Scheraga, 1966), although previous solid-state studies had indicated a /3 structure for poly-n-valine (Blout et al., 1960; Bloom, Fasman, de Laze & Blout, 1962). A tetrapeptide containing a L-Val-L-Val sequence has been suggested possibly to assume the /? conformation in methanol (Shields & McDowell, 1967). These studies further emphasize the important role that solvent takes in determining conformation. Although it has been implied that antiparallel and parallel p conformations have different optical properties in the far ultraviolet region (Pysh, 1966; Rosenheck & Sommer, 1967), a detailed prediction of similarities or differences between the ORD patterns of parallel and antiparallel p structures has not yet been made. Thus a final explanation of our experiments will be contingent upon future theoretical developments. This work was supported in part by grants from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, U.S. Public Health Service (AM 0582), the National Science Foundation (GB 5576) and the U.S. Army Medical Research and Development Command, Department of the Army under Research Contract DA 49193.MD-2933. The paper is Publication no. 690 presented in part at the VII International Biochemistry Congress in Tokyo, August 1967. Also this work was done with the support to one of us (N. M. T.) of a National Institutes of Health training grant (26212) and a Public Health Service fellowship (5-Fl-GM 22,161). It was submitted in partial fulfillment of the requirements for the Ph.D. degree at Brandeis University.
REFERENCES Bird, G. R. t Shurcliff, W. A. (1959). J. Opt. Sot. Amer. 49, 235. Blake, C. C. F., Mair, G. A., North,
A. C. T. t Phillips,
D. C. (1967). Proc. Roy. Sot. B,
167, 356.
Bloom, S. M., Fasman, G. D., de Laze, C. & Blout, E. R. (1962). J. Amer. Chem. Sot. 84, 458. Blout, E. R., de Laze, C., Bloom, S. M. $ Fasman, G. D. (1960). J. Amer. Chem. Sot. 82,
3787. Blout, E. R. & Shechter, E. (1963). Btipolymers,
1, 568. Bohak, Z. t Katchalski, E. (1963). Biochem. 2, 228. Bradbury, E. M., Elliott, A. BEHanby, W. E. (1962). J. Mol. Biol. 5, 487. Cowan, P. M. & McGavin, S. (1955). Nacture, 176, 501. Davidson, B. & Fasman, G. D. (1967). B&hem. 6, 1616. Davidson, B., Tooney, N. t Fasman, G. D. (1966). B&hem. Biophys. Rec. Ccwnm. 23,166. Elliott, A., Fraser, R. D. B., MaoRae, T. P., Stapleton, I. W. & Suzuki, E. (1964). J. Mol. BioZ. 9, 10. Fasman, G. D. (1963). MetWe in Enzymology, 6, 928. Fasman, G. D. & Blout, E. R. (1960). J. Amer. Chem. Sot. 82, 2262. Fasman, G. D., Lindblow, C. & Bodenheimer, E. (1964). Biochem, 3, 155. Fasman, G. D. & Potter, J. (1967). B&hem. Biophys. Res. Comm. 27, 209. Fraser, R. D. B. & MacRae, T. P. (1962). J. Mol. BioZ. 5,467. Gratzer, W. B. t Doty, P. (1963). J. Amer. Chsm. Sot. 85, 1193. Greenfield, N., Davidson, B. & Fasman, G. D. (1967). Biochem. 6, 1630. Huggins, M. L. (1943). Chem. Rewe. 32, 195. Iizuka, E. & Yang, J. T. (1966). Proc. Nat. Acad. Sci., Vaah. 55, 1175. Ikeda, S. t Fasman, G. D. (1967). J. Mol. Bid. 30, 491. Imahori, K. & Yshara, I. (1964). Biopolymers Symp. 1, 421. Jirgensons, B. (1967). J. BioZ. Chem. 242, 912. Johnson, C. (1966). Ph.D. thesis, Massachusetts Institute of Technology. Kendrew, J. C. (1962). Brookhaven Symposium, 15, 216. Kientz, M. L. & Bigelow, C. C. (1966). B&hem. 5, 3494. Lang, C. A. (1968). And. Chem. 80, 1692.
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OF
SERINE
POLYPEPTIDES
369
Makas, A. S. & Shurcliff, W. A. (1955). J. Opt. Sot. Amer. 45, 998. Mitchell, J. C., Woodward, R. B. t Doty, P. (1957). J. Amer. Chem. Sot. 79, 3955. Miyazawa, T. (1960). J. Chem. Phys. 32, 1647. Miyazawa, T. & Blout, E. R. (1961). J. Amer. Chem. Sot. 83, 712. Ooi, T., Scott, R., Vanderkooi, G., Epand, R. F. & Scheraga, H. A. (1966). J. Amer. Chem. Sot. 88, 5680. Parker, K. D. & Rudall, K. M. (1957). Nature, 179, 905. Pauling, L. & Corey, R. B. (1951a). Proc. Nat. Acad. Sci., Wash. 37, 235. Pauling, L. & Corey, R. B. (1951b). Proc. Nut. Accd Sci., Wacrh. 37, 251. Pauling, L. & Corey, R. B. (1953a). Proc. Nat. Acad. Sci., Wash. 39, 253. Pauling, L. & Corey, R. B. (1953b). Proc. Roy. Sot. B, 141, 21. Pysh, E. S. (1966). Proc. Nat. Acad. Sci., Wash. 56, 825. Rosen, H. (1957). Arch. B&hem. Biophys. 67, 10. Rosonheck, K. & Sommer, B. (1967). J. Chem. Phy8. 46, 532. Sage, H. J. & Fasman, G. D. (1966). Biochem. 5, 286. Sarkar, P. & Doty, P. (1966). Proc. Nat. Acad. Sci., Wash. 55, 981. Shields, J. E. & McDowell, S. T. (1967). J. Amer. Chem. Sot. 89, 2499. Tanford, C., Buckley, C. E., De, P. K. & Lively, E. P. (1962). J. Bid. Chem. 237, 1168. Tooney, N. M. & Fasman, G. D. (1968). Riopo~ymers, 6, 81. Yahara, I. & Imahori, K. (1963). J. Amer. Chem. Sot. 85, 230. Yahara, I., Imahori, K., Iitaka, Y. & Tsuboi, M. (1963). J. Polymer Sci. Bl, 47.