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Low temperature circular dichroism of poly (glycyl-l -prolyl-l -alanine)
id. (1969) 39, 307-313
Low temperature Circular Dichroism o Poly (glycyl-L-prolyl-L-alanine) F. R. BROWN, III,J.
P. CARVER? AND E. R. BLQUT
Department of Biological Chemistry Harvard Medical School Bostoq Mass. 02115, U.X.A. (Received 12 August 1968) The circular dichroism of poly(glycyl-L-prolyl-L-danine)$, a synthetic polytripeptide analogue of the non-polar regions of collagen, was investigated in ethylene glycol-water solution (2:1, v/v) over the temperature range of -112°C to + 24°C. Lowering of the temperature results in an increased optical activity of (Gly-Pro-Ala),. At - 112°C (Gly-Pro-Ala), exhibits circular dichroism maxima and minima similar in position, but not as large as those observed for guinea pig skin collagen. A comparison of the low-temperature circular dichroism of (GlyPro-Ala), with that of poly-L-proline II and collagen (in the same solvent and at the same temperature) indicates that the observed increase in optical activity of (Gly-Pro-Ala), is not the result of temperature-dependent solvent effects. Tnus the observed increase in optical activity of (Gly-Pro-Ala), with decreasing temperature is the result of an increase in the amount of periodic structure present in the polymer. Although it is not possible to specify the structure formed in (GlyPro-Ala), at low temperatures in solution, the far ultraviolet circular dichroism is consistent with the structure being a triple helix of the type present in collagen
and suggested for (Gly-Pro-Ala), in the solid state.
1. Introduction In the past many attempts have been made to correlate the proline and/or hydroxyproline content of various collagens with their melting temperatures, T,: in solution (Piez & Gross, 1960; Josse & Harrington, 1964). Recently, Harrington and co-workers 1966) have adapted Schellman’s (1955) (Harrington, 1964; Rao & Herrington, thermodynamic model for the denaturation of proteins to the treatment of the melting temperatures of collagens. The essential step was to include Garrett’s (Flory, 1960) suggestion that the pyrrolidine ring containing residues contribute to the stability of the structured form of collagens by reducing the conformational entropy gain available upon melting of the structure. Consideration of collagen melting temperatures based on this model, and the assumption that collagen is entirely helical, leads to the prediction that both the number and distribution of pyrrolidine residues will affect the melting temperature arrington, 1964; Rao & Harrington, 1966; Carver 8z;Blout, 1967). Because no complete amino acid sequence is available for any collagen, and because there is some question as to which regions, polar, non-polar, or both, are structured in 0 Present address: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, &n&da. $ Abbreviation used in the text for this compound is (Gly-Pro-Ala),. 307
308
F.
R.
BROWN,
III,
J. P. CARVER
AND
E.
R.
BLOUT
collagen, it has been suggested (Carver & Blout, 1967) that this type of treatment could best be evaluated by determining the melting temperatures of synthetic analogs with known sequences resembling those thought to occur in collagen. Ex(a) (Gly-X-Y),, (b) (Gly-Pro-X),, amples of such analogs are the polytripeptides: (c) (Gly-X-Pro),, and (d) (Gly-Pro-Pro),.? These models are expected to have widely different melting temperatures according to various schemes of interchain hydrogen bonding (Table 1). Similarly, considerations of the factors likely to influence the optical activity of collagens led to the suggestion (Carver & Blout, 1967) that the number and distribution of the pyrrolidine ring-containing residues could affect the observed optical activity of collagen and its synthetic analogues. Following considerable effort in several laboratories, some polytripeptides have become available for study. We report here the determination of the far ultraviolet circular diohroism of (Gly-Pro-Ala),, poly-L-proline II, and collagen in ethylene glycol-water solution over the temperature range of -112°C to +24”C.
2. Materials and Methods The sample of (Gly-Pro-Ala), (sample no. RI-98; ?z 2 60) used in the experiments was prepared by the linear polymerization of the tripeptide p-nitrophenyl ester (Bloom, Dasgupta, Pate1 & Blout, 1966). Using this synthesis the resulting polypeptides have a known sequence of amino acid residues, the sequence in the polymer being determined by the tripeptide from which the polymer was prepared. This type of polypeptide having a repeating sequence of identical tripeptide units is termed a “polytripeptide.” The poly (Gly-Pro-Ala) prepared in this fashion and used in these experiments was tested for possible racemization by enzymic hydrolysis. Although no racemization could be detected (Bloom et al., 1966), the possibility of small amounts of racemization could not be ruled out. The collagen sample used in these experiments (JPC-2d) was an acetic acid-extracted preparation of guinea pig skin collagen (method of Gross, 1958). The poly-n-proline (GF-E-29) was prepared from L-prohne N-carboxyanhydride (Fasman & Blout, 1963) and was determined to be authentic poly-L-proline II both by its specific rotation ([cz]~:, = -680” in water) and by its circular dichroism spectrum. The circular dichroism spectra were recorded on a Durrum-Jasco ORD/UV-5 recording spectropolarimeter equipped for operation over the temperature range of -14O’C to +24”C. Low temperatures were obtained with a heat exchanger (AC-110) and vacuum shroud (WMX-1) supplied by Air Products and Chemicals, Inc., Allentown, Pennsylvania. This heat exchanger achieves refrigeration through the expansion of compressed highpressure nitrogen gas ( N 2500 Ib/in2) and it is possible to maintain a temperature stabil&y of + O.l”C. The temperatures at the cell sample holder were measured by a copper versus constantan thermocouple connected to a Honeywell Instruments (model no. 2745) potentiometer. The cells used in the experiments (Opticell, 10.00 to 0.100 mm) exhibited a significant birefringence when optical rota,tory dispersion measurements were attempted at low temperatures. Therefore we report here the results obtained with circular dichroism, where no strain effects of the cells were in evidence. The circular dichroism for (Gly-Pro-Ala),, collagen and poly-L-proline II at -112°C was obtained from samples dissolved in a mixture of ethylene glycol-water (2: 1, v/v). This solvent mixture exists as a fluid glass down to approximately - 140°C; below this temperature it crystallizes sharply. The - 112°C runs were made on samples which had been pre-equilibrated for approximately 48 hr at -30°C and were then immediately cooled down to - 112°C and equilibrated for an additional 4 to 6 hr. The samples were then returned to room temperature ( N 24’C) and allowed to equilibrate for several hours. In all cases the room temperature spectra for samples which had been cooled down and re-equilibrated at room temperature agreed with those spectra obtained before the cooling down process. Thus there appears to be no solvent-induced hysteresis. t X and Y are understood
to be any residue except glycyl,
prolyl,
or hydroxyprolyl
residues.
POLY(GLYCYL-L-PROLYL-L-ALANINE)
3. Results and Discussion Since three defined sequence polytripeptides previously studied, (Gl~r-P~o-~H~)~ (Millionova, 1964; Rogulenkova, Millionova & Andreeva, 1964), (Pro-Gly-Pro), (Engel, Kurtz, Katchalski & Berger, 1966), and (Gly-Pro-Gly), (Oriel $ Blout, 1966) appear to possess some periodic structure in solution, one might expect (Gly-Pro-Ala), also to be regularly structured in solution. However, consideration of the thermodynamics of stabilization in solution of the collagen-like supercoiled triple helical structure (Harrington, 1964) suggests that one would not expect (Gly-Pro-Ala), to be structured at temperatures above approximately -64°C (cf. Carver & Blout, 1967). A previaus report (Oriel & Blout, 1966) did, in fact, show (Gly-Pro-Ala), to be essentially unstructured in aqueous solution at room temperature. The circular dichroism spectrum of (Gly-Pro-Ala), obta,ined at temperatures above and below the expected melting temperature of the polypeptide is shown in Figure 1j In order to compare the spectra obtained at -112°C with those at +24’C, it was necessary to apply a correction factor to the -112°C data. This correction factor arises from a sharpening of the circular dichroism bands and a contraction of the volunle of the solution on cooling. The ma’gnitude of the correct,ion factor was det,ermined by comparing the -112°C and +24”C spectra for a solution of camphor sulfonic acid in ethylene glycol-water (2 : 1, v/v). When t,his correction factor (approximate1.y 13.5% of [P]-112) is ampplied t.o the --112”C data for poly-L-proiine and
1 -61 Ia0
200
220
240
260
Wavelength,A(mp)
1. Circular dichroism curves for (Gly-Pro-Ala),, -). Solvent in both cases, ethykhe glycol-water are corrected for band sharpening and volume contraction I1 FIG.
+24”C (--@--@--) and -112°C (2: 1, v/v). The low-t,emperature dat,a as noted in the text,
310
F.
R.
BROWN,
III,
J. P. CARVER
AND
E. R.
BLOUT
collagen it is found, as shown in Figure 2, that the -112°C and +24”C data agree to within a few per cent. These results indicate that there are no temperature-dependent solvent effects. Therefore the observed increase in optical activity of (Gly-Pro-Ala), with decreasing temperature (Fig. 1) is the result of an increase in the amount of periodic structure present in the polymer. Traub & Yonath (1967) ha.ve recently described two structures for (Gly-Pro-Ala), in the solid state: a collagen type supercoiled triple helix and, in films grown from aqueous solution, a non-supercoiled structure of three helices similar to the collagen I and II structures of Rich & Crick (1961). Both structures have only one systematic hydrogen bond per three residues. This bond involves the NH of the glycyl residue; however, the exact manner of hydrogen bonding is not, as yet, known. At this stage it is not possible to specify the structure formed in (Gly-Pro-Ala), at low temperatures in solution. An agreement between the observed melting temperature for (Gly-Pro-Ala), and that predicted for the polymer in a triple helical conformation would be evidence for (Gly-Pro-Ala), being in a triple helical state. A calculation of T,, assuming AS’P = 3.3 eu (AX&, = 0, AX;,, = 3.9, and AS&,, = 6.0; Rao & Harrington, 1966) and that there is only one hydrogen bond per three residues,
‘r
!
180
I
200
/
/
I
220 Wavelength, .A (mp)
L.-.A-1
240
260
FIG. 2. Circular dichroism curves for poly-L.proline II, +24”C (--O--O--) and -112’C (-O-O-). Circular dichroism curves for collagen, t24”C (--X--X--) and -112’C (-x -X -). Solvent in all cases, ethylene glyool-water (2: 1, v/v). The low-temperature data are corrected for band sharpening and volume contraction as noted in the text. 7 AS” is the mean entropy
change/mole
of peptide
residues upon denaturation.
POLY(GLYCYL-L-PROLYL-r,.ALAiSIXE)
TABLE 1 ~a~c~~te~ and observed melting temperatures (T,) for various ~ol~tr~pe~t~~e models for collagen Polytripept,ide (My-Ma-Ala), (GlyPro-Ala), (My-A!&%-Pro), (I?ro-Gly-Pro),
1500 1500 1800 1500
4.1 2.7 2.7 1.4
-151 -90 -90 +93
-29 -90 193 +93
2070 2070 2070 2070
4.6 3.3 3.3 2.0
-123 -64 -64 +72
t Values assumed by Harrington (1964). $ Values assumed by Rao & Harrington (1966). 5 Calculated for the Rich & Crick (1961) one-bonded structure. ] 1 Calculated for the Ramaohandran (1963) two-bonded structure. Engel et al. (1966) ; (Gly-Pro-Ala),, 7 Observed values: (Pro-Gly-Pro),, temperature over the temperature range of -112’C to +24-T, this work
127 -64 +145 + 72
no defined (see text).
+70
melting
yields T, = -64°C (Table 1). (Gly-Pro-Ala), is not one of the sequences capable of forming -CN* * *O=C hydrogen bonds (Ramachandran & Sasisekharan, 1965), so this value of T, does not contain contributions from these bonds. A melting temperature for the structured form of (Gly-Pro-Ala), in solution is therefore interpretable without recourse to a detailed knowledge of the structure, since one need only know the nature and number of the interchain hydrogen bonds formed. From Figure 3 it can be seen that no sharp transition temperature is evident for (Gly-Pro-Ala), over
!!i g -6.0I-
(b)
-413 Temperature
Fig. 3. Melting profile of (Gly-Pro-Ala), in ethylene circular diohroism maximum at 220 mp; (b) magnitude Both are plotted against temperature (“C).
(“C)
glycol-water (2: 1, v/v). (a) Magnitude :,i’ of circular dichroism minimum at 197 my.
312
F. R.
BROFVN,
III,
J.
P.
CARVER
AK‘U
E:. R.
BLOUT
the temperature range of -112°C to +24”C. The broad “melting” profile (linear rather than sigmoidal dependence of circular dichroism on temperature) may be a result of t,he relatively low molecular weight of the (Gly-Pro-Ala), sample employed in this study (14,500 &500) as compared to collagen (300,000). Engel et al. (1966) have found rather broad melting profiles for (Pro-Gly-Pro), preparations of molecular weight approximately 10,000. We are at present invest’igating a polymer of sequence (Pro-Ala-Gly), synthesized by a coupling of Pro-Ala-Gly tripeptides (Lorenzi & Blout, results to be published). The preparative procedure used minimizes the possibility of racemization (cf. DeTar & Estrin, 1966) and yields a polymer of high molecular weight,. It will be of interest to see how the properties of (Pro-Ala-Gly), compare with (Gly-Pro-Ala), and collagen over a similar temperature range. An additional interesting aspect of the result,s reported in this communication is concerned with the question of adequate models of collagen optical activity. It has been pointed out (Pysh, 1967; Carver & Blout, 1967) that the basic assumptions of degenerate energy levels among monomers required by exciton theory may not be correct for collagen. In particular, the far ultraviolet circular dichroism of collagen can be expected to differ from that of poly-t-proline II for the following reasons: (a) the considerable difference in energy levels between the peptide bond absorptions of pyrrolidine residues and amino acid residues; (b) the absence of a strict repeat in the sequence; (c) tJhe close packing of the three strands to form the collagen triple
I
180
,
200
I
,
220 Wavelength, A Cm/h)
240
I
I
2ko
I!Ic. 4. The --112’C circular dichroism curves for (Gly-Pro-Bla), (--e-e-), poly-L- proline 11 (-O-O-) and collagen (-x - 2 -). Solvent in all cases, ethylene glycol-water (2: 1, v/v). The dat,a are corrected for volume contraction and band sharpening as noted in the text.
l’OLY(GLYCYL-L-PROLYL.~.~L~~I~~)
313
helix; (dj the supercoiling of the individual helices; and (e) the possible existence of regions of the molecule in which there is no periodic structure. It is therefore of interest, to note that the low-temperature far-ultraviolet circular dichroism of (Gly. Pro-Ala), is very simila,r to that of collagen (Fig. 4), whereas that of (Pro-Gly-Pro), is shifted to the red by approximately 5 rnp (Brown, Engel & Blout, unpublished results) and t’hat of poly-L-proline II by approximately 10 rnp.. Simple consideration of the amino acid composition of collagens (~12% prolyl, ~33% glycyl) leads to t.he realization that at maximum there can be only one-third of the triplets in the form -My-Pro-X- or -Gly-X-Pro-, or one-sixt’h in the form -Pro-Gly-Pro-. Since (Gly-ProAla), is a model only for the so-called non-polar regions of collagen, it is not expected that the circular dichroism spectrum of this polymer would be necessarily ident’ieal wit,h that of collagen. In view of our knowledge of the amino acid composition of eollagens, at least two-thirds of the molecule should consist of -(Gly-X-Y)- triplets. It will be of considerable interest, therefore, to see how the far ultraviolet circular dichroism of (Gly-Ala-Ala), compares with that of (Gly-Pro-Ala), and collagen itself. \Ye are pleased to acknowledge the support in part of this work by G.S. Pubhc He&h Service grant AM-07300. One of us (F.R.B.) is a National Science Foundation Predoctorai Yellow; another (J.P.C.) is a Helen Hay Whitney Postdoct,oral Fellow (1966-68). REFERENCES Bloom, S. M., Dasgupta, S. K.: Patel, R. P. & Bloat, E. R. (1966). J. --1~,er. Chem. Sot 88, 2035. C’ar\-er, J. P. & Blout, E. R. (1967). In Treatise OWLCoZZuye~~, ed. by G. S. Ramachandran, vol. 1, p. 441. London: Academic Press. Ikfi’ar, ID. F. & E&in, N. F. (1966). Tetruhedmn Letters, 110. 48, 5985. Engel: J., Rurtz, J.. Katchalski, E. Bi Berger, A. (1966). J. illol. Biol. 17. 266. Fasman, G. D. & Blorrt, E. R. (1963). Riopolymers, 1, 3. Glory? P. J. (1960). Brookhaven Synap. Biol. 13, 230. Gross, J. (1958). J. Ezp. Med. 107, 247. Biol. 9, 613. Barrington, TV. I?. (1964). J. Mol. Jesse, J. & Narriugton, LT. F. (1964). J. Mol. BioZ. 9, 269. Xillionova, M. I. (1964). Biophysics, 9, 149. Oriel, P. J. & Blout, E. R. (1966). J. Amer. Chem. Sot. 88, 2041. Piez, Ii. A. & Cross, J. (1960). J. Biol. Chew. 235, 995. Pysh, 4~. S. (1967). J. Mol. Biol. 23, 687. R,amachandran, C. N. (1963). In Aspects of I-‘rotein Stmctwe, od. by G. S. R#amachandran, p. 39. London: Academic Press. Ramachandran, G. S. & Sasisekharan, V. (1965). Biochim. biophys. A&, 109, 314. R,ao, hr. V. & Narrington, W. F. (1966). J. Mol. Biol. 21, 577. Rich, A. & Crick: F. H. C. (1961). J. Mol. BioZ. 3, 483. R,ogulenkova, V. PI’., Millionova, M. I. & Andreeva, N. 8. (1964). J. ,%loZ. Biol. 9, 253. Sehellman, J. A. (1956). C. R. Lab. Carlsberg, Sir. Cizitn.. 29, 230. Traub> M’. & Vonath, A. (1967). J. Mol. BioZ. 25, 3.51.
Low temperature Circular Dichroism o Poly (glycyl-L-prolyl-L-alanine) F. R. BROWN, III,J.
P. CARVER? AND E. R. BLQUT
Department of Biological Chemistry Harvard Medical School Bostoq Mass. 02115, U.X.A. (Received 12 August 1968) The circular dichroism of poly(glycyl-L-prolyl-L-danine)$, a synthetic polytripeptide analogue of the non-polar regions of collagen, was investigated in ethylene glycol-water solution (2:1, v/v) over the temperature range of -112°C to + 24°C. Lowering of the temperature results in an increased optical activity of (Gly-Pro-Ala),. At - 112°C (Gly-Pro-Ala), exhibits circular dichroism maxima and minima similar in position, but not as large as those observed for guinea pig skin collagen. A comparison of the low-temperature circular dichroism of (GlyPro-Ala), with that of poly-L-proline II and collagen (in the same solvent and at the same temperature) indicates that the observed increase in optical activity of (Gly-Pro-Ala), is not the result of temperature-dependent solvent effects. Tnus the observed increase in optical activity of (Gly-Pro-Ala), with decreasing temperature is the result of an increase in the amount of periodic structure present in the polymer. Although it is not possible to specify the structure formed in (GlyPro-Ala), at low temperatures in solution, the far ultraviolet circular dichroism is consistent with the structure being a triple helix of the type present in collagen
and suggested for (Gly-Pro-Ala), in the solid state.
1. Introduction In the past many attempts have been made to correlate the proline and/or hydroxyproline content of various collagens with their melting temperatures, T,: in solution (Piez & Gross, 1960; Josse & Harrington, 1964). Recently, Harrington and co-workers 1966) have adapted Schellman’s (1955) (Harrington, 1964; Rao & Herrington, thermodynamic model for the denaturation of proteins to the treatment of the melting temperatures of collagens. The essential step was to include Garrett’s (Flory, 1960) suggestion that the pyrrolidine ring containing residues contribute to the stability of the structured form of collagens by reducing the conformational entropy gain available upon melting of the structure. Consideration of collagen melting temperatures based on this model, and the assumption that collagen is entirely helical, leads to the prediction that both the number and distribution of pyrrolidine residues will affect the melting temperature arrington, 1964; Rao & Harrington, 1966; Carver 8z;Blout, 1967). Because no complete amino acid sequence is available for any collagen, and because there is some question as to which regions, polar, non-polar, or both, are structured in 0 Present address: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, &n&da. $ Abbreviation used in the text for this compound is (Gly-Pro-Ala),. 307
308
F.
R.
BROWN,
III,
J. P. CARVER
AND
E.
R.
BLOUT
collagen, it has been suggested (Carver & Blout, 1967) that this type of treatment could best be evaluated by determining the melting temperatures of synthetic analogs with known sequences resembling those thought to occur in collagen. Ex(a) (Gly-X-Y),, (b) (Gly-Pro-X),, amples of such analogs are the polytripeptides: (c) (Gly-X-Pro),, and (d) (Gly-Pro-Pro),.? These models are expected to have widely different melting temperatures according to various schemes of interchain hydrogen bonding (Table 1). Similarly, considerations of the factors likely to influence the optical activity of collagens led to the suggestion (Carver & Blout, 1967) that the number and distribution of the pyrrolidine ring-containing residues could affect the observed optical activity of collagen and its synthetic analogues. Following considerable effort in several laboratories, some polytripeptides have become available for study. We report here the determination of the far ultraviolet circular diohroism of (Gly-Pro-Ala),, poly-L-proline II, and collagen in ethylene glycol-water solution over the temperature range of -112°C to +24”C.
2. Materials and Methods The sample of (Gly-Pro-Ala), (sample no. RI-98; ?z 2 60) used in the experiments was prepared by the linear polymerization of the tripeptide p-nitrophenyl ester (Bloom, Dasgupta, Pate1 & Blout, 1966). Using this synthesis the resulting polypeptides have a known sequence of amino acid residues, the sequence in the polymer being determined by the tripeptide from which the polymer was prepared. This type of polypeptide having a repeating sequence of identical tripeptide units is termed a “polytripeptide.” The poly (Gly-Pro-Ala) prepared in this fashion and used in these experiments was tested for possible racemization by enzymic hydrolysis. Although no racemization could be detected (Bloom et al., 1966), the possibility of small amounts of racemization could not be ruled out. The collagen sample used in these experiments (JPC-2d) was an acetic acid-extracted preparation of guinea pig skin collagen (method of Gross, 1958). The poly-n-proline (GF-E-29) was prepared from L-prohne N-carboxyanhydride (Fasman & Blout, 1963) and was determined to be authentic poly-L-proline II both by its specific rotation ([cz]~:, = -680” in water) and by its circular dichroism spectrum. The circular dichroism spectra were recorded on a Durrum-Jasco ORD/UV-5 recording spectropolarimeter equipped for operation over the temperature range of -14O’C to +24”C. Low temperatures were obtained with a heat exchanger (AC-110) and vacuum shroud (WMX-1) supplied by Air Products and Chemicals, Inc., Allentown, Pennsylvania. This heat exchanger achieves refrigeration through the expansion of compressed highpressure nitrogen gas ( N 2500 Ib/in2) and it is possible to maintain a temperature stabil&y of + O.l”C. The temperatures at the cell sample holder were measured by a copper versus constantan thermocouple connected to a Honeywell Instruments (model no. 2745) potentiometer. The cells used in the experiments (Opticell, 10.00 to 0.100 mm) exhibited a significant birefringence when optical rota,tory dispersion measurements were attempted at low temperatures. Therefore we report here the results obtained with circular dichroism, where no strain effects of the cells were in evidence. The circular dichroism for (Gly-Pro-Ala),, collagen and poly-L-proline II at -112°C was obtained from samples dissolved in a mixture of ethylene glycol-water (2: 1, v/v). This solvent mixture exists as a fluid glass down to approximately - 140°C; below this temperature it crystallizes sharply. The - 112°C runs were made on samples which had been pre-equilibrated for approximately 48 hr at -30°C and were then immediately cooled down to - 112°C and equilibrated for an additional 4 to 6 hr. The samples were then returned to room temperature ( N 24’C) and allowed to equilibrate for several hours. In all cases the room temperature spectra for samples which had been cooled down and re-equilibrated at room temperature agreed with those spectra obtained before the cooling down process. Thus there appears to be no solvent-induced hysteresis. t X and Y are understood
to be any residue except glycyl,
prolyl,
or hydroxyprolyl
residues.
POLY(GLYCYL-L-PROLYL-L-ALANINE)
3. Results and Discussion Since three defined sequence polytripeptides previously studied, (Gl~r-P~o-~H~)~ (Millionova, 1964; Rogulenkova, Millionova & Andreeva, 1964), (Pro-Gly-Pro), (Engel, Kurtz, Katchalski & Berger, 1966), and (Gly-Pro-Gly), (Oriel $ Blout, 1966) appear to possess some periodic structure in solution, one might expect (Gly-Pro-Ala), also to be regularly structured in solution. However, consideration of the thermodynamics of stabilization in solution of the collagen-like supercoiled triple helical structure (Harrington, 1964) suggests that one would not expect (Gly-Pro-Ala), to be structured at temperatures above approximately -64°C (cf. Carver & Blout, 1967). A previaus report (Oriel & Blout, 1966) did, in fact, show (Gly-Pro-Ala), to be essentially unstructured in aqueous solution at room temperature. The circular dichroism spectrum of (Gly-Pro-Ala), obta,ined at temperatures above and below the expected melting temperature of the polypeptide is shown in Figure 1j In order to compare the spectra obtained at -112°C with those at +24’C, it was necessary to apply a correction factor to the -112°C data. This correction factor arises from a sharpening of the circular dichroism bands and a contraction of the volunle of the solution on cooling. The ma’gnitude of the correct,ion factor was det,ermined by comparing the -112°C and +24”C spectra for a solution of camphor sulfonic acid in ethylene glycol-water (2 : 1, v/v). When t,his correction factor (approximate1.y 13.5% of [P]-112) is ampplied t.o the --112”C data for poly-L-proiine and
1 -61 Ia0
200
220
240
260
Wavelength,A(mp)
1. Circular dichroism curves for (Gly-Pro-Ala),, -). Solvent in both cases, ethykhe glycol-water are corrected for band sharpening and volume contraction I1 FIG.
+24”C (--@--@--) and -112°C (2: 1, v/v). The low-t,emperature dat,a as noted in the text,
310
F.
R.
BROWN,
III,
J. P. CARVER
AND
E. R.
BLOUT
collagen it is found, as shown in Figure 2, that the -112°C and +24”C data agree to within a few per cent. These results indicate that there are no temperature-dependent solvent effects. Therefore the observed increase in optical activity of (Gly-Pro-Ala), with decreasing temperature (Fig. 1) is the result of an increase in the amount of periodic structure present in the polymer. Traub & Yonath (1967) ha.ve recently described two structures for (Gly-Pro-Ala), in the solid state: a collagen type supercoiled triple helix and, in films grown from aqueous solution, a non-supercoiled structure of three helices similar to the collagen I and II structures of Rich & Crick (1961). Both structures have only one systematic hydrogen bond per three residues. This bond involves the NH of the glycyl residue; however, the exact manner of hydrogen bonding is not, as yet, known. At this stage it is not possible to specify the structure formed in (Gly-Pro-Ala), at low temperatures in solution. An agreement between the observed melting temperature for (Gly-Pro-Ala), and that predicted for the polymer in a triple helical conformation would be evidence for (Gly-Pro-Ala), being in a triple helical state. A calculation of T,, assuming AS’P = 3.3 eu (AX&, = 0, AX;,, = 3.9, and AS&,, = 6.0; Rao & Harrington, 1966) and that there is only one hydrogen bond per three residues,
‘r
!
180
I
200
/
/
I
220 Wavelength, .A (mp)
L.-.A-1
240
260
FIG. 2. Circular dichroism curves for poly-L.proline II, +24”C (--O--O--) and -112’C (-O-O-). Circular dichroism curves for collagen, t24”C (--X--X--) and -112’C (-x -X -). Solvent in all cases, ethylene glyool-water (2: 1, v/v). The low-temperature data are corrected for band sharpening and volume contraction as noted in the text. 7 AS” is the mean entropy
change/mole
of peptide
residues upon denaturation.
POLY(GLYCYL-L-PROLYL-r,.ALAiSIXE)
TABLE 1 ~a~c~~te~ and observed melting temperatures (T,) for various ~ol~tr~pe~t~~e models for collagen Polytripept,ide (My-Ma-Ala), (GlyPro-Ala), (My-A!&%-Pro), (I?ro-Gly-Pro),
1500 1500 1800 1500
4.1 2.7 2.7 1.4
-151 -90 -90 +93
-29 -90 193 +93
2070 2070 2070 2070
4.6 3.3 3.3 2.0
-123 -64 -64 +72
t Values assumed by Harrington (1964). $ Values assumed by Rao & Harrington (1966). 5 Calculated for the Rich & Crick (1961) one-bonded structure. ] 1 Calculated for the Ramaohandran (1963) two-bonded structure. Engel et al. (1966) ; (Gly-Pro-Ala),, 7 Observed values: (Pro-Gly-Pro),, temperature over the temperature range of -112’C to +24-T, this work
127 -64 +145 + 72
no defined (see text).
+70
melting
yields T, = -64°C (Table 1). (Gly-Pro-Ala), is not one of the sequences capable of forming -CN* * *O=C hydrogen bonds (Ramachandran & Sasisekharan, 1965), so this value of T, does not contain contributions from these bonds. A melting temperature for the structured form of (Gly-Pro-Ala), in solution is therefore interpretable without recourse to a detailed knowledge of the structure, since one need only know the nature and number of the interchain hydrogen bonds formed. From Figure 3 it can be seen that no sharp transition temperature is evident for (Gly-Pro-Ala), over
!!i g -6.0I-
(b)
-413 Temperature
Fig. 3. Melting profile of (Gly-Pro-Ala), in ethylene circular diohroism maximum at 220 mp; (b) magnitude Both are plotted against temperature (“C).
(“C)
glycol-water (2: 1, v/v). (a) Magnitude :,i’ of circular dichroism minimum at 197 my.
312
F. R.
BROFVN,
III,
J.
P.
CARVER
AK‘U
E:. R.
BLOUT
the temperature range of -112°C to +24”C. The broad “melting” profile (linear rather than sigmoidal dependence of circular dichroism on temperature) may be a result of t,he relatively low molecular weight of the (Gly-Pro-Ala), sample employed in this study (14,500 &500) as compared to collagen (300,000). Engel et al. (1966) have found rather broad melting profiles for (Pro-Gly-Pro), preparations of molecular weight approximately 10,000. We are at present invest’igating a polymer of sequence (Pro-Ala-Gly), synthesized by a coupling of Pro-Ala-Gly tripeptides (Lorenzi & Blout, results to be published). The preparative procedure used minimizes the possibility of racemization (cf. DeTar & Estrin, 1966) and yields a polymer of high molecular weight,. It will be of interest to see how the properties of (Pro-Ala-Gly), compare with (Gly-Pro-Ala), and collagen over a similar temperature range. An additional interesting aspect of the result,s reported in this communication is concerned with the question of adequate models of collagen optical activity. It has been pointed out (Pysh, 1967; Carver & Blout, 1967) that the basic assumptions of degenerate energy levels among monomers required by exciton theory may not be correct for collagen. In particular, the far ultraviolet circular dichroism of collagen can be expected to differ from that of poly-t-proline II for the following reasons: (a) the considerable difference in energy levels between the peptide bond absorptions of pyrrolidine residues and amino acid residues; (b) the absence of a strict repeat in the sequence; (c) tJhe close packing of the three strands to form the collagen triple
I
180
,
200
I
,
220 Wavelength, A Cm/h)
240
I
I
2ko
I!Ic. 4. The --112’C circular dichroism curves for (Gly-Pro-Bla), (--e-e-), poly-L- proline 11 (-O-O-) and collagen (-x - 2 -). Solvent in all cases, ethylene glycol-water (2: 1, v/v). The dat,a are corrected for volume contraction and band sharpening as noted in the text.
l’OLY(GLYCYL-L-PROLYL.~.~L~~I~~)
313
helix; (dj the supercoiling of the individual helices; and (e) the possible existence of regions of the molecule in which there is no periodic structure. It is therefore of interest, to note that the low-temperature far-ultraviolet circular dichroism of (Gly. Pro-Ala), is very simila,r to that of collagen (Fig. 4), whereas that of (Pro-Gly-Pro), is shifted to the red by approximately 5 rnp (Brown, Engel & Blout, unpublished results) and t’hat of poly-L-proline II by approximately 10 rnp.. Simple consideration of the amino acid composition of collagens (~12% prolyl, ~33% glycyl) leads to t.he realization that at maximum there can be only one-third of the triplets in the form -My-Pro-X- or -Gly-X-Pro-, or one-sixt’h in the form -Pro-Gly-Pro-. Since (Gly-ProAla), is a model only for the so-called non-polar regions of collagen, it is not expected that the circular dichroism spectrum of this polymer would be necessarily ident’ieal wit,h that of collagen. In view of our knowledge of the amino acid composition of eollagens, at least two-thirds of the molecule should consist of -(Gly-X-Y)- triplets. It will be of considerable interest, therefore, to see how the far ultraviolet circular dichroism of (Gly-Ala-Ala), compares with that of (Gly-Pro-Ala), and collagen itself. \Ye are pleased to acknowledge the support in part of this work by G.S. Pubhc He&h Service grant AM-07300. One of us (F.R.B.) is a National Science Foundation Predoctorai Yellow; another (J.P.C.) is a Helen Hay Whitney Postdoct,oral Fellow (1966-68). REFERENCES Bloom, S. M., Dasgupta, S. K.: Patel, R. P. & Bloat, E. R. (1966). J. --1~,er. Chem. Sot 88, 2035. C’ar\-er, J. P. & Blout, E. R. (1967). In Treatise OWLCoZZuye~~, ed. by G. S. Ramachandran, vol. 1, p. 441. London: Academic Press. Ikfi’ar, ID. F. & E&in, N. F. (1966). Tetruhedmn Letters, 110. 48, 5985. Engel: J., Rurtz, J.. Katchalski, E. Bi Berger, A. (1966). J. illol. Biol. 17. 266. Fasman, G. D. & Blorrt, E. R. (1963). Riopolymers, 1, 3. Glory? P. J. (1960). Brookhaven Synap. Biol. 13, 230. Gross, J. (1958). J. Ezp. Med. 107, 247. Biol. 9, 613. Barrington, TV. I?. (1964). J. Mol. Jesse, J. & Narriugton, LT. F. (1964). J. Mol. BioZ. 9, 269. Xillionova, M. I. (1964). Biophysics, 9, 149. Oriel, P. J. & Blout, E. R. (1966). J. Amer. Chem. Sot. 88, 2041. Piez, Ii. A. & Cross, J. (1960). J. Biol. Chew. 235, 995. Pysh, 4~. S. (1967). J. Mol. Biol. 23, 687. R,amachandran, C. N. (1963). In Aspects of I-‘rotein Stmctwe, od. by G. S. R#amachandran, p. 39. London: Academic Press. Ramachandran, G. S. & Sasisekharan, V. (1965). Biochim. biophys. A&, 109, 314. R,ao, hr. V. & Narrington, W. F. (1966). J. Mol. Biol. 21, 577. Rich, A. & Crick: F. H. C. (1961). J. Mol. BioZ. 3, 483. R,ogulenkova, V. PI’., Millionova, M. I. & Andreeva, N. 8. (1964). J. ,%loZ. Biol. 9, 253. Sehellman, J. A. (1956). C. R. Lab. Carlsberg, Sir. Cizitn.. 29, 230. Traub> M’. & Vonath, A. (1967). J. Mol. BioZ. 25, 3.51.