the triple helix ⇌ coil conversion of collagen-like polytripeptides in aqueous and nonaqueous...

BIOPOLYMERS VOL. 16, 601-622 (1977)

The Triple Helix 6 Coil Conversion of Collagen- Like Polytripeptides in Aqueous and Nonaqueous

Solvents. Comparison of the Thermodynamic Parameters and the Binding of Water to (L-Pro-L-

Pro-Gly) and (L-Pro-L-Hyp-Gly)

JURGEN ENGEL, HAN-TEE CHEN, and DARWIN J. PROCKOP, Department of Biochemistry, Rutgers Medical School, College of

Medicine and Dentistry of N e w Jersey, Piscataway, New Jersey 08854, and HORST KLUMP, Znstitut f u r Physikalische Chemie, Uniuersitiit

Freiburg, Freiburg, Wes t Germany

Synopsis

The collagen-like peptides (r,-Pro-r,-Pro-Gly), and (L-Pro-1.-Hyp-Gly), with n = 5 and 10, were examined in terms of their triple helix +coil transitions in aqueous and nonaqueous solvents. The peptides were soluble in 1,2-propanediol containing 3% acetic acid and they were found to form triple-helical structures in this solvent system. The water content of the solvent system and the amount of water bound to the peptides were assayed by equilibrating the solvent with molecular sieves and carrying out Karl Fischer titrations on the solvent phase. After the solvent was dehydrated, much less than one molecule of water per tripeptide unit was bound to the peptides. Since the peptides remained in a triple-helical conformation, the results indicated that water was not an essential component of the triple-helical structure. Comparison of peptides with the same chain length demonstrated that the presence of hydroxyproline increased the thermal stability of the triple helix even under anhydrous conditions. The results, therefore, did not support recent hypotheses that hydroxyproline stabilizes the triple helix of collagen and collagen-like peptides by a specific interaction with water molecules. Analysis of the thermal transition curves in several solvent systems showed that although the peptides containing hydroxyproline had t , values which were 18.6' to 32.7"C higher, the effect of hydroxyproline on AG was only 0.1 to 0.3 kcal per tripeptide unit a t 25°C. The results suggested, therefore, that the influence of hydroxyproline on helical stability may he explained by intrinsic effects such as dipole-dipole interactions or by changes in the solvation of the peptides by alcohol, acetic acid, and water. A direct calorimetric measurement ofthe transition enthalpy for (IA-Pro-r,-Pro-Gly), in 3% or 1OOh acetic acid gave a value of -1.84 kcal per tripeptide unit for the coil-to-helix transition. From this value for enthalpy and from data on the effects of different chain lengths on the thermal transition, it was calculated that the apparent free energy for nucleation was +5 kcal/mol at 25°C (apparent nucleation parameter = 2 X lo-" M-". The value was dependent on solvent and on chemical modification of end groups.

INTRODUCTION

The collagen triple helix consists of three peptide chains each of which is folded into a polyproline-11-like helix with about three residues per

60 1

ci 1977 by John Wiley & Sons, Inc.

602 ENGEL ET AL.

t ~ r n . l - ~ One necessary condition for formation of the triple helix is that every third amino acid in each chain is glycine and that each chain consists of repeating -Gly-X-Y- sequencces. Because glycine does not have a carbon side chain, it can be positioned in the interior of the triple helix at each turn of the helix, and hydrogen bonds can form between the peptide NH of glycine and the peptide CO of an amino acid residue which is in the X- position of an adjacent helix. The three helices are, therefore, held in intimate contact. The side chains of the amino acids in the X- and Y- positions point away from the central axis of the triple helix, and as a result most amino acids can be readily accommodated in these positions.

Formation of the triple helix is also favored by the presence of proline and hydroxyproline in the X- and Y-positions of the three chains, since restriction of rotation around the $ and II/ angles of these imino acids pro- motes folding of each chain into a polyproline I1 helix. The ready rotation around the $ and II/ angles of glycine, however, prevents individual chains from forming separate helical structures even in the case of synthetic peptides rich in imino acids such as (Gly-L-Pro-L-Pro), or (Gly-L-Pro- L-HYP),. The triple helix of collagen must, therefore, be considered as a unique structure which forms directly from three coiled

A third condition which favors formation of the triple helix is the presence of hydroxyproline. Collagen is one of the few proteins in nature which contains hydroxyproline and this hydroxy imino acid is present in about 100 of the Y-positions of the 330 repeating -Gly-X-Y- tripeptide units found in each chain of most collagens from vertebrate^.^^^ About two decades ago G u s t a v ~ o n ~ , ~ suggested that hydroxyproline might help to stabilize collagen fibers by providing additional sites for hydrogen-bond formation among adjacent molecules. Gustavson's hypothesis has not yet been substantiated (see Refs. 5 and 9), but in the past several years hydroxyproline has been shown to stabilize the formation of the triple helix itself. A form of collagen without hydroxyproline was isolated from connective tissue cells and shown to form a triple-helical structure at low tempera- t u r e ~ . ~ - ~ ~ The helical structure, however, had a thermal transition about 15°C lower than the thermal transition of collagen containing hydroxyproline. A similar effect was observed with two cyanogen bromide peptides from collagen which differed only in their hydroxyproline ~ 0 n t e n t . l ~ In addition, hydroxyproline was shown to stabilize the triple helix formed by synthetic peptides resembling collagen in that (L-Pro-L-Hyp-Gly)lo in aqueous solution had a thermal transition about 25°C higher than the thermal transition of (L-Pro-L-Pro-Gly)

Although glycine, proline, and hydroxyproline play major roles in formation and stabilization of the collagen triple helix, it seems likely that formation of the collagen helix is also favored by less obvious effects such as interactions among amino acid side groups in adjacent chains of the triple helix,15 and by interactions of the polypeptides with solvent. The im- portance of such effects is suggested by comparisons of collagens from different animals. Collagens from such sources differ markedly in their

(L-PRO-L-PRO-GLY)n AND (L-PRO-L-HYP-GLY)n 603

thermal stability and it is difficult to account for these differences solely on the basis of differences among the proteins in their contents of glycine, proline, and hydroxyproline. 1 6 3 1 7

In the present report we have examined the thermodynamic properties of the triple helix + coil transition of synthetic peptides with repeating sequences of -L-Pro-L-Pro-Gly- or -L-Pro-L-Hyp-Gly- in alcohols and mixtures of alcohols and water. These studies were combined with measurements of water binding and designed to check a recent hypothesis advanced in two different forms by Ramachandran et a1.2 and by Traub4 that hydroxyproline stabilizes the triple helix by hydrogen bonding to a water molecule which becomes incorporated into the helical structure.

MATERIALS AND METHODS

All polytripeptides were synthesized for us by Dr. S. Sakakibara and his coworkers, Protein Research Foundation, Minoh-Shi, Osaka, Japan. The polytripeptides were prepared by specific modifications of the Merrifield technique for solid-phase synthesis.14,18 1,2-Propanediol, U.S.P., was obtained from Baker Chemical Co., and was stored over molecular sieves. A sample was vacuum distilled but there was no improvement in transparency. Glacial acetic acid and methanol were Baker analyzed reagents. The reagents for the Karl Fischer titration were purchased from Fisher Scientific Co. Water calibrations were performed with sodium tartrate certified A.C.S. from Fisher Scientific Co.

The molecular sieves 3 A were purchased from Grace, Davidson Chem- ical, Baltimore, Md. The beads were extensively washed with methanol in order to remove dust and they were dried a t 250°C for 1 hr before use. For the Karl Fischer titrations the titration assembly TTAl/KF was employed in combination with the polarizing adapter L 409/100 KQ, the ti- trator TTTll and the pH meter PHM26, all from Radiometer, Copenha- gen.

The circular dichroism (CD) was recorded with a Cary Model 61 spec- tropolarimeter (Varian, Monrovia, Calif.). Thermostated cells of 1-mm path length (Opticell Co., Beltsville, Md.) were used. For High-temperature work above 9O"C, a cell was modified in the following way. A heating coil (about 5 turns, resistance about 5 Q) was placed around the outer diameter of the cell, which was filled with silicon oil for better thermal con- ductivity. The cell was placed on a patch of asbestos and fixed on a wooden support which replaced the metal prism in the cell holder of the instrument. A thermistor (glass bead, 1 mm diameter, 2 KR a t 25°C) was attached to the outer surface by a heat-conducting silicone paste (from GC Electronics, Rockford, Ill.). This thermistor was used for regulating the current through the heating coil by an on-off controller (Model 63RC, Yellow Springs In- strument Co., Yellow Springs, Ohio). The current was regulated with an autotransformer. Typical settings were 10 V, 2 A a t 100°C and 15 V, 3 A at 180°C. The temperature was measured between the windows in the

604 ENGEL ET AL.

upper part of the sample compartment 1 mm above the light beam by a calibrated thermistor of diameter 0.8 mm which was permanently attached to the teflon stopper of the cell. The temperature constancy which was achieved was better than f0.2"C in the range of 50" to 190°C.

Calorimetric measurements were performed with the help of a newly developed differential adiabatic calorimeterlg which is suitable for the investigations of thermally induced conformational transitions of dissolved polymers. Heats effects of 250 mcal can be measured with a precision of f5% referred to a temperature interval of 20°C and 25 ml of solution. The heating rate was about 0.23"C/min. The suitable temperature range is -10" to +llO°C. The molecular weights were determined by the sedi- mentation equilibrium technique in a Spinco Model E analytical ultra- centrifuge (Beckman Instruments, Paolo Alto, Calif.). To calculate molecular weights of the peptides in 1,2-propanediol, the partial specific volume was assumed to be 0.71, a value which seems reasonable on the basis of previous observations20 with poly(L-proline) in aqueous and nonaqueous solvents.

MECHANISM AND EVALUATION OF THE THERMODYNAMIC DATA

The simplest mechanism which has been proposed21,22 for the coil * triple-helix transition may be summarized by the scheme:

in which C is a randomly coiled chain, Hi is a helical species with i tripeptide units in a triple-helical conformation, P is the cooperativity parameter for the nucleation step, Ps" is the equilibrium constant for the formation of a nucleus H,, and s is the equilibrium constant for steps following nucleation. The nucleus H, is defined as the first product for which the elongation of the triple helix is faster than its dissociation. A characteristic feature of a tripeptide in the triple-helical conformation is the presence of a hydrogen bond between the NH group of glycine and the CO of proline in an adjacent chain. Since the chains are staggered by one amino acid residue and the tripeptides a t the extreme ends of the triple helix cannot form hydrogen bonds, three chains containing n tripeptide units can form a maximum of 3n - 2 hydrogen bonds, and the complete triple helix is designated as H3n-2.

The number of helical tripeptide units in the nucleus H , has been estimated on the basis of kinetic data to be either six21 or eighteen.22 After formation of the nucleus, the triple helix is completed via propagation steps and in each of these steps a coiled tripeptide unit adjacent to an existing stretch of helix becomes helical. Since the propagation steps are essentially identical, each of them is assigned the same equilibrium constant. The process is simplified when n is relatively small, and P and the total concentration of chains, c , are small so that pc2 is less than one.21p22 Under

(L-PRO-L-PRO-GLY), AND (L-PRO-L-HYP-GLY), 605

these conditions, the concentration of intermediate species [Hi] becomes small compared to [C] and [H:3n-2]. The formation of helix then becomes an "all-or-none'' reaction and the reaction can be described by the overall equilibrium constant.

where F is the degree of helicity in terms of the fraction of chains or tripeptide units of helical state, and

F = (3 ) 3[H:<n-2]

C

From

AG" = -RT In K = AH" - TAS" (4)

and from Eq. (2) it follows that a t the midpoint of the transition ( F = 0.5 and T = T,,,),

(5)

where AGO is the standard free energy (Gibbs free energy), AH" is the standard enthalpy, and AS" is the standard entropy. I t should be noted that T,,, is concentration dependent and only values which are measured at identical molar peptide concentrations, or which are corrected by Eq. (5) to a common concentration, can be used for a comparison of stability.

AH" is usually obtained from the slope of the transition curves and therefore with much less precision than T,. A first approximation of AH" can be obtained from the Van't Hoff equation

AH" AS" + R In ( 0 . 7 5 ~ ~ )

T,,, =

AH" = 8RTm2 (") dT F = 0.5

It is generally more accurate, however, to evaluate AH" by curve fitting to the entire transition curve with the equation

AH" T RT T ,

K = exp [ ~ (- - 1) - ln 0.75c2] ( 7 )

which is obtained by solving Eq. ( 5 ) for AS" and substituting for AS" in Eq. (4). Values of K obtained with Eq. (7) can be used to calculate F with Eq. (2). To obtain the data presented here, a first estimate of AH" was obtained from Eq. (6) and this value of AH" was used to calculate F a t a series of temperatures by using Eqs. (7) and (2). The calculated values of F were compared to observed values of F , and curve fitting was then carried out to obtain the best value of AH".

Values for AH" can also be obtained by direct calorimetric measurements. Calorimetric measures of AHo are generally more accurate and

606 ENGEL ET AL.

they provide a test for the reaction mechanism. If the calorimetric AHo is about the same as the Van't Hoff AHo estimated from thermal transition curves (see above), the reaction probably proceeds by an all-or-none mechanism as described in Eq. (1) and it probably does not involve intermediate species which contain both helical and coiled regions.24

If AHo and T, are known, ASo can be calculated from Eq. (5). The values of AH" and AS" can then be used to calculate AGO at any temperature by the relationship AGO = AHo - TASO. Alternatively, AGO can be estimated by using Eq. (2) to calculate K from observed values of F , and then using the relationship AGO = - R T In K. Estimates of AGO, however, are most accurately obtained at temperatures near the T,, since K cannot be accurately estimated by Eq. (2) for values of F which approach 0 or 1.0, and since errors in AH" and A S o tend to cancel each other only near Tnl.

EXPERIMENTAL RESULTS

Water Content of the Polytripeptides

(L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), in the solid state always contain some ~ a t e r . ~ ~ J ~ , ~ ~ - ~ ~ The exact amount depends on the water content of the air with which the peptides are in equilibrium but the last molecule of water per tripeptide unit (about 6 wt %) cannot be removed by extensive drying over P205.14

In order to examine the peptides under reproducible conditions, we equilibrated them at 25°C in a nonevacuated desiccator in which a constant humidity of 75% was maintained with a saturated solution of sodium chlorate. Because 75% humidity was close to that of the laboratory at- mosphere, no change of weight because of a loss or uptake of water was observed while weighing the samples. The water contents were determined by Karl Fischer titration or by quantitative amino acid analysis (see Methods). The calculation of water contents from amino acid analysis depends on the assumption that the samples contain no salts or materials other than peptide and water. As indicated in Table I, satisfactory

TABLE I Water Contents Expressed in Weight Percent for the

Polytripeptides Equilibrated at 75% Relative Humidity and 25°C

Water Content from Amino Acid Peptide Karl Fischer Titrationa Analysisb

( L-Pro-L-Pro-Gly ), ,, 15.5 f 3.5 15.9 ( L-Pro-L-Pro-Gly ), 24 f 0.4 22.4 (L-Pro-L-Hyp-Gly),, 14.6 c 1.8 8.6 (L-Pro-L-Hyp-Gly ), 15.3 f 3.2 10.1

a Averages of two to four independent determinations performed at intervals

b Single determinations. of several months. The errors indicated are standard deviations.

(L-PRO-L-PRO-GLY)n AND (L-PRO-L-HYP-GLY), 607

Fig. 1. Water-binding isotherm for the molecular sieve zeolite 3 A in 1,2-propanediol/acetic acid at 25°C. Capacity (G) indicates milligrams of water bound per milligram of the molecular sieve: concentration of water is the solvent water (?$( ) in equilibrium with the molecular sieve.

agreement between the two methods was obtained. Because it was more reliable and direct, the Karl Fischer method was used in most of the experiments reported here.

Binding of Water to the Polytripeptides in 1,2-Propanediol

The binding of water to polytripeptides dissolved in 1,2-propanediol was assayed by using a solvent system equilibrated with zeolite molecular sieves (see Methods). The molecular sieves served to establish a known concentration of solvent water according to their binding isotherm. Karl Fischer titrations were used first to measure the solvent water when the molecular sieve was equilibrated with the solvent alone, and then to measure the sum of the solvent water plus peptide-bound water when the molecular sieve was equilibrated with solvent containing a known concentration of polytripeptide.

As a first step in the assay, a binding isotherm was established for the zeolite molecular sieve. The weight fraction of water bound to the molecular sieve, or “capacity” of the molecular sieve (G) is

- u(c, - c,)

z ( l - C,/lOOO) G =

where u is the initial volume of the mixture of 1,2-propanediol and water in milliliters, z is the weight of the molecular sieve in milligrams, c,, is the total concentration of water in the system and EU is the concentration of solvent water where all concentrations are expressed as milligram/milliliter. The factor (1 - C,/10001 corrects for the small change in volume of the solvent phase which occurs when the molecular sieve is added and absorbs water from the system. The binding isotherm (Fig. 1) was obtained by carrying out Karl Fischer titrations on solvent mixtures of 1,2-propane-

608 ENGEL ET AL.

diol/water before and after the solvent mixtures were equilibrated with the molecular sieve.

It might be noted that the approach employed here assumes that only water will penetrate the molecular sieve. This assumption is not critical but it is reasonable since the pore size of the molecular sieve is 3 A, the critical diameter of water is 3.2 A, and the critical diameter of 1,2-propanediol is over 5 A. Also, no measurable change in volume was observed when up to 1 ml of water was added to 10 g of zeolite equilibrated with 10 ml of 1,2-propanediol.

After the binding isotherm was developed (Fig. 11, the binding of water to the polytripeptides was measured by using the relationship

u ( c w - r ) z ( l - r/1000)

G =

where r is the sum of the concentrations of solvent water and of the water bound to the peptide. The value of r was directly determined by the Karl Fischer titration of water in the solvent phase. The value for cw was obtained from the sum of the total water of the system before addition of the peptide plus the amount of water added with the peptide (Table I). With these values, Eq. (9) was used to calculate G , and the calculated value of G was used to estimate Fw from the binding isotherm (Fig. 1). The concentration of peptide-bound water was then calculated as r - C,. The molar fraction of water bound per tripeptide, f , was calculated as

where M,. is the molecular weight of the tripeptide unit and cp is the concentration of peptide in milligram/milliliter.

In the experiments carried out here, the polytripeptides (L-Pro-L-Pro- Gly)S, (L-Pro-L-Pro-Gly)lo, and (L-Pro-L-Hyp-Gly)lo were dissolved in 1,2-propanediol in a concentration of 5 to 10 mg/ml. About 1.6 ml of the solution was equilibrated with about loo0 mg of zeolite for 48 hr after which r was measured by the Karl Fischer titration. The concentration of solvent water was adjusted to the desired values by addition of measured amounts of water before equilibration. Values off were obtained with satisfactory precision only at low concentrations of solvent water. The error was estimated from the reproducibility of the titrations and the reproducibility of the isotherm. At C, = 0.15 mgjml, the error off was *lo%. At higher values of CU, the error increased, because the concentration of peptide-bound water had to be calculated as the difference between two relatively large numbers. A t Fw = 0.7 mg/ml, the error was f40%, and this was about the highest concentration of solvent water a t which meaningful values for f could be obtained.

The results (Fig. 2) demonstrated that a t moderately low values of CuI, considerably less than one molecule of water was bound per tripeptide unit of either (L-Pro-L-Pro-Gly), or (L-Pro-L-Hyp-Gly),. From the data, it


lor-

0 02 04 06 0 8 10 CONCENTRATION OF WATER [ "'g/,l]

Fig. 2. Molar fraction of water per tripeptide unit vs. equilibrium concentration of solvent water in 1,2-propanediol/acetic acid a t 25°C. 0 (L-Pro-l,-Pro-Gly),,,, 0 (I,-Pro-L-Pro-GIy)>, 0 (I.-Pro-I~-Hyp-Gly),,,, and (~-Pro-~,-Hyp-Gly)r,. The dotted curve is calculated for the hinding of water per tripeptide unit with a binding constant K = 5 M - ' . This is the highest value which is consistent with the data.

appears that the binding constant ( K = 18 f / F [ , , ) cannot be greater than 5 M-1.

Comparison of Helix + Coil Transition of (L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), in Different Solvent Systems

A solvent in which the transitions of (L-Pro-L-Pro-Gly), and (L-Pro- ~-Hyp-Gly), can be compared must fulfill a number of requirements. 1) Both peptides must be soluble in the solvent. 2) The helix-to-coil transition must take place in a temperature range in which the solvent does not boil or freeze. 3) The solvent must be transparent enough to permit CD measurements near 225 nm.

Preliminary experiments demonstrated that 1,2-propanediol/acetic acid (97:3 v/v) satisfied these three conditions. The peptides were sparingly soluble in 1,2-propanediol itself, but adequate concentrations were obtained when 3% acetic acid was added. Since 1,2-propanediol is liquid from -30" to 198"C, it was possible to use this solvent to examine the thermal transitions of all four peptides. Also, although the solvent system was not transparent below about 225 nm, it was possible to follow the ellipticity of the two conformations with a good signal-to-noise ratio a t 237.5 nm (see below). In addition, the peptides were shown by equilibrium ultracen- trifugation to form triple helical structures in 1,2-propanediol/acetic acid at 20°C. The observed molecular weight for (L-Pro-L-Pro-Gly)," was 7300 (calculated molecular weight for three chains, 7600) and the observed molecular weight for (L-Pro-L-Hyp-Gly)lo was 7900 (calculated, 8100).

MethanoUacetic acid 9O:lO (v/v) was also satisfactory in terms of solu- bility and transparency, but because it boils at 64"C, only (L-Pro-L-Pro- Gly), and (L-Pro-L-Hyp-Gly)F, could be examined in this solvent. Aqueous acetic acid (3% v/v) was only suitable for (L-Pro-L-Pro-Gly)lo and (L- Pro-L-Hyp-Gly) because the thermal transitions of the shorter peptides were at or below the freezing point of the solvent.

Most of the measurements reported here were performed with peptide concentrations between 1 and 2 mM. A t concentrations less than 1 mM,

610 ENGEL ET AL.

equilibrium developed too slowly to be measured accurately in the early part of the transition curves. For each experiment, the peptides were equilibrated at low temperatures for 24 hr in order to allow complete helix formation and the temperature was then increased in intervals of 5" to 10°C. Equilibrium was generally achieved within 20 to 60 min near the midpoint of the transition but much longer times were needed at F > 0.7 [Eq. (3)]. The transition curves were followed as far as possible into the regions where the peptides are entirely in helical or coiled conformation, since an accurate estimate of the effect of temperature on the ellipticity of these two forms is required for calculation of the thermodynamic parameters.

The transition curves of (L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), were compared in three solvent systems (see Figs. 3,4, and 5). The molar ellipticity of the helical conformations was found to be independent of temperature (see Fig. 3(b) and Fig. 5). In contrast, the ellipticity of the randomly coiled forms showed a pronounced temperature dependence in all solvent systems and this was approximated by a linear dependence with a slope d[O]/dT = q. In 1,2-propanediol/acetic acid, q could only be determined accurately for ( L - P ~ o - L - P ~ o - G ~ ~ ) ~ and (~-Pro-~-Hyp-Gly)5 [Fig. 3(a)], and at 237.5 nm identical slopes of q = -7.2 (deg cm2/K dmol) were found. A similar slope was approximated with the limited data for (L- Pro-L-Hyp-Gly)lo and (L-Pro-L-Pro-Gly)10 [Fig. 3(b)] and q = -7.2 was therefore used for all four peptides in this solvent. In 3% aqueous acetic acid, the q value for (L-Pro-L-Pro-Gly)lo was -10.8 (Fig. 5). The coiled form of (L-Pro-L-Hyp-Gly),o differed, however, in that the molecular ellipticity was much less negative than in 1,2-propanediol and the value for q was only -4.8.

The thermodynamic parameters were evaluated in the following way. The temperature dependencies of the molar ellipticities of the pure states were extrapolated over the transition range. The melting temperature T , was then measured as the point at which the distances between the transition curve and the extrapolated lines were equal. Designating the ellipticity observed at any temperature as {& the ellipticity of the coiled form as (6lc at T,, and the ellipticity of the helix as ( 0 ) h at T,, the degree of conversion F [Eq. (3)] a t any temperature becomes

Since T , is known, AH" is the only parameter which has to be varied in the curve-fitting procedure [Eq. (7)]. For the transition curves in methanol/acetic acid (Fig. 4), q was also varied since its direct evaluation was not possible because of the low boiling point of methanol. The most probable value appeared to be q = -15.6 {deg cm2/K dmol), a relatively high value which is probably explained by the low wavelength ( A = 235 nm) used to measure the transition. When q was assumed to be zero for methanol/ acetic acid, the A€€" values were about 15% less negative but the T,,, was


r

-20:o b io 40 60 80 IbO I20 I i O I60 l8( TEMPERATURE ("C)

(b 1 Fig. 3. Temperature-induced equilibrium transition curves of (1.-Pro-L-Hyp-Gly), and

(i.-Pro-r.-Pro-Gly),, in 1,2-propanediol/acetic acid 9?:3 (v/v) as followed by molar ellipticity {/)I a t 237.5 nm. (a) n = 5, c = 2.1 m M for (L-Pro-L-Hyp-Gly)s and 2.3 mM for (L-Pro-1,- Pro-Gly),. (b) n = 10, c = 2.3 mM for (L-Pro-L-Hyp-Gly)lO and 2.2 mM for (L-Pro-L-Pro- Gly) IO. Filled circles: measured at rising temperature; open circles: measured at decreasing temperature. Theoretical transition curves were calculated using the following parameters. (i.-Pro-i,-Hyp-Gly)R: T, = 335.?'K, AHo = -35 kcal/mol; (L-Pro-L-Pro-Gly)s: T, = 310.2'K, A H o = -21 kcal/mol; (L-Pro-L-Hyp-Gly)IO: T, = 404.2OK, A H o = -50 kcal/mol; and (I~-Pro-r,-Pro-Gly),": T, = 385.2'K, AHo = -45 kcal/mol. For (L-Pro-L-Hyp-Gly)lc and (L-Pro-L-Pro-GIy),0 theoretical curves with 20% higher and 20% lower A H o values are also calculated [dashed curves in (b)]. The slope of the temperature dependence of the ellipticity of the coiled form is q = -3.6 deg cm2/K dmol (dashed lines).

612 ENGEL ET AL.

16

12

8

-- 4 43 3 0 N

k? x .-4 I c

-8

-I 2

-16

HYP

0 20 40 60 TEMPERATURE ( 'C )

Fig. 4. Temperature-induced equilibrium transition curves for (L-Pro-L-Hyp-Gly)s (c = 0.48 mM) and (L-Pro-L-Pro-Gly)s (c = 0.72 mM) in methanol/acetic acid 9 0 1 0 (v/v). The theoretical curves are calculated with the following parameters. (L-Pro-L-Hyp-Gly)s: T, = 317.9'K, AH' = -40 kcal/mol; (L-Pro-L-Pro-Gly)S: T, = 297.7'K, AHo = -30 kcal/mol. The slope q is assumed to be -7.8 deg cm2/K drnol (dashed line).

n *\

-*\ -2

-6

-8 0 20 40 60 80

TEMPERATURE ("C)

-8 0 20 40 60 80

TEMPERATURE ("C)

Fig. 5. Temperature-induced equilibrium transition curves for(L-Pro-I,-Hyp-Gly) 1 0 (c = 2.4 mM) and (L-Pro-L-Pro-Gly),o (c = 2.6 mM) in water/acetic acid 97:3 (v/v). Theoretical curves are calculated with the following parameters. (L-Pro-L-Hyp-Gly)lo: T, = 334.7'K, A H o = -90 kcal/mol; (L-Pro-L-Pro-Gly)lO: T, = 304.2'K, AHo = -53 kcal/mol. The slopes y are -5.4 for (L-Pro-L-Pro-Gly)lo and -2.4 for (L-Pro-L-Hyp-Gly)lo, respectively (dashed lines).


TABLE I1 Thermodynamic Data for the Coil + Triple-Helix Transition

of ( L-Pro-L-Hyp-Gly ), and ( L-Pro-L-Pro-Gly ), (n = 5 and 10) in different solvent systems

T m > LW", ASo, AG;,, AAG:,, At,,,, Peptide Solvent "Ka kcal/molb e.u./rnolb kcal/molb kcal/molc "C"

~ ~_______

(L-Pro-L-Hyp-Gly ),, ( L -Pro-L -Pro-Gly ) ~

( L-Pro-L-Hyp-Gly), ( L -Pro- L -Pro-Gly ) I

( L -Pro- L -Hy p-Gly ) ( L-Pro-L-Pro-Gly ),

(L-Pro-L-Hyp-Gly), (L-Pro-L Pro-Gly),,

1,2-propanediol/ 393.8 -50 -".O -20'5 -0.103 18.6 acetic acid- 375.2 -45 -91.9 -17.6 97:3 (v /v) 326.5 -35 -79.2 -11.4

methanol/ 325.4 -40 -94.9 -11.7

-0.25 30.8 295.7 -21 -43 -8.18

acetic acid- 301.6 -30 -71.4 -8.70 -0.23 23.8 9O:lO (viv)

water/ 330.5 -90 -244 -17.1 acetic acid- 297.8 -53 -150.6 -8.29 32'7 97:3 (v lv) -51.5d

a For c = 1 mM. b Expressed per triple helix. c AAG = AG(L-Pro-L-Hyp-G1y)-AG( L-Pro-L-Pro-Gly) expressed per tripeptide

d Determined calorimetrically. unit.

essentially the same. Since AH" was obtained essentially from the slope of the curves and since the slopes were very much influenced by experimental errors, the accuracy of this determination was rather low. This is demonstrated by Figure 3(b) in which the theoretical transition curves for 20% higher and 20% lower AH" values were compared with the transition curve which gave the best fit. With this approach it was estimated that the error for determining AH was f15%. Because the error of AGO was much lower (f5%) the error of AS" is about as large as that of AH" but the two errors are not independent [see Eq. (4)].

For better comparison, the T,,, values which were measured at peptide concentrations between c = 0.5 and 2 mM are corrected to c = 1 mM by Eq. (5). AH", ASo, and AGO are expressed per mole of triple helix. The difference in free energy between peptides containing hydroxyproline and those without hydroxyproline ( AAG ") is, however, expressed per tripeptide unit in order to allow a better comparison of results obtained with peptides of different chain length. The difference between the melting temperatures of (L- Pro-L-Hyp-Gly), and (L-Pro-I>-Pro-Gly), are given in the last column of Table 11.

The thermodynamic data are summarized in Table 11.

Calorimetric Determinations of AH"

Measurements were performed with (L-Pro-L-Pro-Gly) 10 and (L-Pro- I,-Pro-Gly)ls in both 3% and 10% aqueous acetic acid, because (L-Pro-1,- Pro-Gly)ls was not sufficiently soluble in 3% acetic acid. The measured profiles of compensating energies required to maintain adiabatic condition versus temperature are shown in Figure 6. The rate a t which the temperature was increased in the calorimeter was too high (13 deg/h) to es-

614 ENGEL ET AL.

Fig. 6.

XI

10 and 15) in aqueous acetic acid. The compensating energy which is needed to maintain adiabatic conditions is plotted vs. temperature. - - - - : (L-Pro-L-Pro-Gly)10, c = 1.3 mM, and (L-Pro- i,-Pro-Gly),>, c = 0.916 mM, in water/acetic acid 9010 v/v; -: (L-Pro-L-Pro-Gly),o, c = 1.4 mM in water/acetic acid 97:3 (v/v). The sample volume was 25 ml.

tablish equilibrium in the part of the transition curve for which F > 0.5. As a result, the peaks were distorted and the half-conversion temperatures taken from the calorimetric transition curves were higher than T,. The temperature at which the peaks merged with the baseline on the high- temperature side was, however, in good agreement with the corresponding points in the equilibrium transition curve. The AHo values were calculated from the areas under the curves in Figure 6 and are summarized in Table 111. Unfortunately calorimetric determinations of AH" for (L-Pro-L- Hyp-Gly), were not possible because of lack of sufficient amounts of these polymers. Measurements in 1,Z-propanediol were also excluded because it was impossible to work at temperatures higher than 80°C with the instrument available.

Thermodynamic Parameters in Mixtures of 1,2-Propanediol and Water

It was of interest to see how the thermodynamic parameters of (L-Pro- L-Pro-Gly), and (L-Pro-L-Hyp-Gly), changed in mixtures of the alcohols and water. Unfortunately it was not possible to perform such measurements for (L-Pro-L-Hyp-Gly)lo, since this peptide has a high-melting

TABLE I11 Calorimetrically Determined AHo Values

aH", kcal/mol Peptide Solvent peptide

10% aqueous acetic acid -4r1.5 ( L -Pro-L -Pro-Gly ), 3% aqueous acetic acid -51.5

(L-Pro-L-Pro-Gly), 10% aqueous acetic acid -75.0 ( L -Pro-L -Pro-G1 y ),


10

30

Fig. 7. Temperature-induced transition of (r,-Pro-L-Pro-Gly),,) in mixtures of 1,Z-propanediol/acetic acid and water at 25°C as followed by CD. The transition was followed a t the following ratios of water to l,Z-propanediol/acetic acid (viv): 1OO:O ( c = 1.78 mM): 0 ; 5050 ( c = 2.27 mM): v; 27:73 (c = 2.54 mM): A; 15:85 ( c = 2.57 mM): 0; and 0:lOO ( c = 2.2 mM): 0. Filled symbols: measurements a t increasing temperature; open symbols: measurements a t decreasing temperature. The theoretical curves were obtained by a best f i t to the experimental data using the AH" and ASo values which are plotted in Figure 8.

temperature in water and the melting temperature is raised to above the boiling point of the solvent when 1,2-propanediol is added. With (L-Pro- r,-Hyp-Gly)s and (L-Pro-L-Pro-Gly)S, it was not possible to carry out the measurements with water contents of more than 20% and 10%, respectively, because the thermal transitions became too low. Figure 7 shows the thermal transition curves of (L-Pro-L-Pro-Gly) 10 a t various solvent com- positions. Both the CD signal for the helical state and fer the coiled state of the chains became smaller with increasing water content, but the position of the positive maximum in the CD spectrum a t 225 nm did not shift and the temperature dependence of the CD signal of the coiled form seemed to be independent of solvent composition. The thermodynamic quantities were obtained by a fit of theoretical transition curves to the experimental data (Fig. 7) as described in the preceding paragraphs, and they are plotted versus solvent composition in Figure 8 together with similar but more limited data for (L-Pro-L-Hyp-Gly)S. The AGO values for (L-Pro-L-Pro- Gly)r, were obtained from a measurement of the solvent-induced transition at constant temperature.

As indicated (Fig. 8), a minimum in A H o and ASo, but not in AGO, was observed with about 20% water in 1,2-propanediol. The initial change in A H o as the 1,2-propanediol concentration was increased was consistent with the fact that hydrogen bonds were being transferred from water to a less polar solvent. A similar effect on AHo was observed with (L-Pro-L- Pro-Gly)S in mixtures of water and n-propanol (unpublished results).

616 ENGEL ET AL.

Or------

~ 1250'6 02 04 0.6 0.8 1 0 '

VOLUME FRACTIONOFWATER IN 1.2- PROPANEDIOL

Fig. 8. Dependence of AGISr AH0, and AS0 for (L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), on the volume fraction of water in 1,2-propanediol. 0-0: (L-Pro-L-Pro-Gly)s; m-¤ (L- Pro-I.-Hyp-Gly)s; 0-0 (I~-Pro-L-Pro-Gly)lo; and 0-0 (L-Pro-L-Hyp-Gly)lo.

Chain-Length Dependence of the Thermodynamic Data

The mechanism outlined in Eq. (1) suggests that AG", AH", and ASo should change by constant increments with increasing chain length because of the contribution of additional propagation steps, as long as 3n - 2 > x and as long as the all-or-none approximation [Eq. (2)] is valid for all chain lengths. We can write therefore:

AG" = AG; + (3n - 2)AGi

AH" = AH; + (3n - 2)AGi

ASo = ASK + (3n - 2)ASi

where AG:, AH:, and AS: are the thermodynamic quantities which are associated with the equilibrium constant of propagation. An extrapolation of these linear dependencies to 3n - 2 = 0 yields the apparent nucleation parameters AG;, AH;, and AS; which are associated with the nucleation step, with end effects, or other effects which occur only once per triple- helical molecule.

Figure 9 shows the thermodynamic parameters of (L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), plotted according to Eq. (12). For (L-Pro-L-


0 - al 0 . -

2 -50 r I

I a -100 !-

0

- : -100 E . 3

? -200 cn a

-300 0 10 20 30 40 50 60

3n-2

Fig. 9. Plot of the thermodynamic parameters AG;:, A H o , and A S o vs. the maximum number of tripeptide units which participate in the triple-helix formation in peptides of chain length n. A: (I,-Pro-L-Pro-Gly), in water/acetic acid 9O:lO (v/v); 0: (L-Pro-L-Pro-Gly)lo in water/acetic acid 97:3 (v/v); 0: (IL-Pro-L-Pro-Gly), in 1,2-propanediol/acetic acid 97:3 (v/v); V: (r,-Pro-I.-Hyp-Gly),, in 1,2-propanediol/acetic acid 97:3 (v/v). For comparison, the graphs include the data of Kobayashi et a1.;2H 0: (t,-Pro-L-Pro-Gly),, in water/acetic acid 9010 (v/v) and by Sutoh and N ~ d a ; ~ ~ X: (L-Pro-I,-Pro-Gly), in water/acetic acid 97:3 (v/v). The data of Kobayashi et al.'"and of Sutoh and N ~ d a ' ~ have been recalculated on the basis of the calorimetrically determined AHo values (see Table I11 and text).

Pro-Gly), in aqueous acetic acid only calorimetric enthalpies were used because of their high accuracy, and because an accurate value is important for the extrapolation to 3n - 2 = 0. The data published by Kobayashi et a1.28 and by Sutoh and NodaZ9 are also included in Figure 9. Instead of using the original data which were based on Van't Hoff enthalpies, AG" and AS" values were calculated from the published T, values and the calorimetric enthalpies obtained here. The large discrepancies which orig- inally existed among the data in the l i t e r a t ~ r e , ~ ~ . ~ ~ - ~ O and between these data and our own, vanished when all the calculations were carried out with the calorimetrically determined enthalpies. Figure 9 shows that there is an excellent agreement among the data from different sources and that there is little or no difference between values measured in 3% or 10% aqueous acetic acid. For (L-Pro-L-Pro-Gly), in dilute acetic acid, AG; is +5 kcal and AG% = -0.49 kcal a t 25°C. The corresponding enthalpies and entropies are AH; = 0 and AH: = -1.85 kcal, AS; = -17 e.u., and AS:

618 ENGEL ET AL.

= -4.5 e.u., when AG;, AH;, and AS; are expressed per mole triple helix and AG;, AH:, and AS: were expressed per mole tripeptide unit.

The values for chain-length dependence in 1,2-propanediol are less accurate than the values in aqueous acetic acid since the only data available were for n = 5 and 10 and only the Van’t Hoff enthalpies were known. It is apparent however that solvent had a large influence on the apparent nucleation parameters.

DISCUSSION

Because the peptides (L-Pro-L-Pro-Gly), and (L-Pro-L-Hyp-Gly), are soluble in several different solvents, it was possible to develop information as to how the triple-helical structures formed by the peptides are influenced by three factors: the binding of water, the presence of the 4-hydroxyl group in hydroxyproline, and the length of the peptide chain.

The Binding of Water to the Triple Helix

The results demonstrated that there was little if any water bound to either the peptides, which contained hydroxyproline, or to those which did not contain hydroxyproline when the peptides were dissolved in 1,2-propanediol containing 3% acetic acid. From the binding data (Fig. 2) it was apparent that essentially no water was bound to the peptides when they were dissolved in 1,2-propanediol in which the final water concentration was 1% (v/v) or less. After the 1,2-propanediol/acetic acid was dried with molecular sieves, its water content was less than 0.01%. Nevertheless, the peptides formed triple-helical structures in this solvent system. The presence of water is, therefore, not an essential part of the triple-helical structure formed by either (L-Pro-L-Pro-Gly), or (L-Pro-L-Hyp-Gly),.

The conclusion that water is not an essential part of the triple helix was supported by the experiments in which the water content of the solvent system was varied (Fig. 8). The free energy of the coil-to-helix transition became less negative when water was added and for all peptides the triple helix was less stable in water than in alcohol. Moreover, the change in free energy for the coil-to-helix transition of (L-Pro-L-Pro-Gly)lo, (L-Pro-L- Pro-Gly),, and (~-Pro-~-Hyp-Gly), was smooth as increasing amounts of water were added to the alcohol. If water had a specific stabilizing effect and if water was strongly bound to the helical structure, addition of a small amount of water to the peptides should have abruptly changed the stability of the helix.

It should be noted that the low affinity of peptides for water was some- what unexpected, since it was previously observed that both (L-Pro-L- Pro-Gly), and (L-Pr-L-Hyp-Gly), retain one mole of water per tripeptide unit after being dried in uucuo over P205 at 80°C for 22 hr.*4,27 The present data, however, do not contradict the earlier observations for several reasons. One reason is that when the peptides are dried, binding sites for water may


be formed among adjacent molecules, and such binding sites may disappear when the peptides are solubilized. A second reason is that complete drying of a peptide may be difficult because complete removal of water may create "voids" in the structure which give rise to large, unsatisfied intermolecular forces.31 A third consideration is that when the peptides are dissolved in 1,2-propanediol/acetic acid, the binding of water to the peptides will be affected by competing interactions of water with the alcohol and by competing interactions of the alcohol with the peptides. The binding for water observed here may differ considerably from the binding which might occur if the peptides were in an aqueous environment. Unfortunately, there are serious theoretical and practical difficulties in obtaining binding parameters for water to peptides in aqueous solution (see Ref. 31).

Effect of the 4-Hydroxyl Group of Hydroxyproline on Helical Stability

Several different models have been advanced to explain why the presence of 4-hydroxyproline stabilizes the triple-helical structure formed by collagen. Ramachandran et aL2 pointed out that a water molecule which they located as forming an interchain bridge in their model for collagen was in a favorable position to form a third hydrogen to the 4-hydroxyl group of hydroxyproline and thereby help to stabilize the helix further. Traub4 proposed a variant on his model for (Gly-Pro-Hyp), in which a water molecule forms a hydrogen-bonded bridge between the 4-hydroxyl of hydroxyproline and the carbonyl of the preceding glycine in the same chain. Berg et al.32 developed a model for the triple helix formed by (Pro-Hyp- Gly),, in which they proposed that the 4-hydroxyl group on hydroxyproline formed a direct hydrogen bond with a carbonyl group in an adjacent chain. Bansal et al."" recently examined this proposed structure by model building using computer techniques and found it comparable in stability with other models from energy considerations.

One of the critical observations made here was that the helical structure of (L-Pro-L-Hyp-Gly), had a higher thermal stability than the triple helix of (L-Pro-L-Pro-Gly), when both peptides were dissolved in 1,2-propanediollacetic acid under conditions in which the amount of water bound to either peptide was negligible. The results demonstrated, therefore, that the stabilizing effect of hydroxyproline on the triple-helical structures formed by the peptides in 1,2-propanediollacetic acid cannot be explained by any model which assumes a specific binding of water to the 4-hydroxyl group. Such water bridges may form in aqueous solutions, but on the basis of the data obtained here it must be concluded that if a bridging of the OH groups of hydroxyproline with CO groups of the peptide has a stabilizing effect this effect will be rather unspecific and may be also ex- hibited by 1,2-propanediol, methanol or acetic acid.

It should be noted, however, that the data also do not provide support for the proposal that the 4-hydroxyl group forms a direct hydrogen bond

620 ENGEL ET AL.

with an adjacent chain.3z Preliminary experiments suggested that the presence of hydroxyproline changed the AH" for the coil-to-helix transition by about -2 kcal/mol of tripeptide unit, a value which is consistent with the formation of an additional hydrogen bond. The more extended measurement carried out here demonstrated that although the presence of hydroxyproline changed the AH" by -1.3 kcal/mol of tripeptide unit in watedacetic acid, the effect was -0.7 in methanol/acetic acid and only -0.2 kcal/mol of tripeptide unit for (L-Pro-L-Hyp-Gly)lo and (L-Pro-L-Pro- Gly) 10 in 1,2-propanediol/acetic acid (Table 11). There is still considerable uncertainty about the AH0 value one can ascribe to a hydrogen bond and 1,2-propanediol may have some unusual influence on the' AH" of collagen-like peptides. The effect of hydroxyproline on AH" observed here, however, appears too small to be consistent with the suggestion that the presence of the 4-hydroxyl group allows the formation of an additional hydrogen bond.

Since the influence of hydroxyproline on AH" and AGO was relatively small, it may be that the 4-hydroxyl group stabilizes the helix by an intrinsic effect such as providing a dipole which enhances favorable electrostatic interactions. Alternatively, the presence of hydroxyproline may enhance solvation of the triple helix. In support of this possibility, it may be noted that with both peptides the AH0 for the coil-to-helix transition was more negative in water than in the organic solvents but this helix-favoring AH0 was more than compensated by the AS" becoming more negative. As a result, both helical structures became less stable in water. However, the effect of water on both AH" and AS" was greater for (L-Pro-L-Hyp-Gly), than for (L-Pro-L-Pro-Gly),. The direction and magnitude of these effects suggests that in aqueous solutions the triple helix formed by both types of peptides interacts with water but the helix formed by peptides containing hydroxyproline is preferentially hydrated. Some of the water in the in- nermost solvation shell may, in fact, fit into the structure with the orien- tation of a water bridge from the 4-hydroxyl to other functional groups as suggested by model-building s t u d i e ~ . ~ , ~ In anhydrous solvents such as 1,2-propanediol/acetic acid the water is readily displaced but the new solvent interactions may again be more extensive in the case of the helical structure formed from peptides containing hydroxyproline. (One of the referees of this paper pointed out in support of this view that it can be shown by space-filling models that the OH-group in COOH of acetic acid or at carbon 1 of 1,2-propanediol may also bridge between the OH-group of hydroxyproline and the CO-group of the collagen chain.)

Effect of Chain Length

The effect of chain length on the helical stability of (L-Pro-L-Pro-Gly), has now been examined by a number of i n v e s t i g a t ~ r s . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Most of the studies have been carried out in 3% or 10% aqueous acetic acid, and the values for the melting temperatures reported by different authors are


similar. There are, however, relatively large discrepancies in the thermodynamic parameters, particularly in the values for the Van’t Hoff AHo. As a result, there has been considerable uncertainty as to the value of AG; and the nucleation parameter, p = exp (AGh/RT). Published values of 6 range from 3 x MP2 29 to 5 x loP3 M-2 28 for (L-Pro-L-Pro-Gly), in dilute acetic acid. These discrepancies are probably explained by the large error of f15% in calculating Van’t Hoff AHo from thermal transition curves. In contrast, the error of the calorimetric measurements carried out here is only f5%. When our own data and previously published data were calculated by using the calorimetric value for enthalpy, most of the discrepancies were resolved, and the results indicated that the extrapolated value for AGh was +5 kcal/mol of triple helix.

The large dependence of AG; or on solvent indicates that end effects play an important role. In aqueous solution, the ends are charged and probably repulse each other when in the helical structure.. Evidence for such repulsion was obtained by titration of the imino-terminal group of (L-Pro-L-Pro-Gly) 10 in the helical state, and the observation that the thermal transition decreases around the isoelectric point of the ~ e p t i d e . ~ ~ Repulsion of charged end groups is also consistent with the large negative ASo which is seen in aqueous solution, since accumulation of charges might produce an ordering of water around the ends of the peptides. In anhydrous alcohols, the end groups are not ionized and this may explain the more negative AGk,. An effect of end groups is also suggested by the observation that the AGL for the protected peptides Z(G1y-L-Pro-L-Pro), OBut is more positive (8.8 kcal/mol):35 in methanol/water than the AG; for the unpro- tected peptides.

If, as the results suggest, helix formation by the peptides is influenced by end groups, it will probably be necessary to reconsider the conclusions of several previous studies in which end effects were neglected and exper- imentally determined values for AGk and p were assumed to correspond to the nucleation p a r a m e t e r ~ . ~ ~ , ~ ~ , ~ ~ J ~ For example, it is probably not possible to estimate the cooperative length of the triple helix from the apparent nucleation parameter which was obtained by examining the effect of chain length in the all-or-none region.24 Also, it is probably incorrect to calculate the rate constant of helix propagation from the observed overall rate constant for helix formation and the observed value for p.21,29 In the all-or-none case, the AGO for the nucleation process and the AGO for end effects are additive. Therefore, it is unlikely that the AGO for the nucleation process can be estimated separately unless measurements on the coil-to-helix transition are carried out with peptides which have chain lengths ranging from the cooperative length of the triple helix to well above the cooperative length. Until more accurate values of AGO for the nucleation process are available, it will be difficult to evaluate kinetic models for the coil-to-helix transition.

This work was supported in part by Research Grant AM-16,516 from the National Institutes of Health of the U. S. Public Health Service.

622 ENGEL ET AL.

References

1. Ramachandran, G. N. (1967) in Treatise on Collagen, Ramachandran, G. N., Ed.,

2. Ramachandran, G. N., Bansal, M. & Bhatnagar, R. S. (1973) Biochim. Biophys. Acta

3. Traub, W., Yonath, A. & Segal, D. M. (1969) Nature 221,914-917. 4. Traub, W. (1974) Israel J. Chem. 12,435-439. 5. Traub, W. & Piez, K. (1971) Aduan. Protein Chem. 25,243-341. 6. Fietzek, P. & Kuhn, K. (1976) Int . Reu. Connective Tissue 7, l . 7. Gustavson, K. H. (1954) Acta Chem. Scand. 8,1298-1305. 8. Gustavson, K. H. (1955) Nature 175,70-74. 9. Prockop, D. J., Berg, R. A,, Kivirikko, K. I. & Uitto, J . (1976) in Biochemistry of Col-

10. Uitto, J . & Prockop, D. J. (1973) Abstracts 9th Int. Congr. Biochemistry (Stockholm),

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13. Ward, A. R. & Mason, P. (1973) J. Mol. Biol. 79,431-435. 14. Sakakibara, S., Inouye, K., Shudo, K., Kishida, Y., Kobayashi, Y. & Prockop, D. J . (1973)

15. Salem, G. & Traub, W. (1975) FEBS Letters 51,94-97. 16. Privalov, P. L. & Tiktopulo, E. J. (1970) Biopolymers 9, 127-139. 17. Cooper, A. (1971) J. Mol. Biol. 55, 123-127. 18. Sakakibara, S., Kishida, Y., Kikucha, Y., Sakai, R. & Kikiuchi, K. (1968) Bull. Chem.

19. Gruber, M. & Ackermann, Th. (1974) Z. Phys. Chem. (Neue Folge) 93,255-264. 20. Knof, S. & Engel, J. (1974) Israel J . Chem. 12,165-177. 21. Weidner, H., Engel, J. & Fietzek, P. (1974) in Peptides, Polypeptides and Proteins,

Blout, E. R., Bovey, F. A., Goodman, M. & Lotan, N., Eds., Wiley, New York, pp. 419-435. 22. Sutoh, K. & Noda, H. (1974) Biopolymers 13,2477-2488. 23. Go, N. & Suezeki, Y. (1973) Biopolymers 12,1927-1930. 24. Engel, J . & Schwarz, G. (1970) Angew. Chem. 82,468-479; Angew. Chem., Int. Ed. 9,

25. Yonath, A. & Traub, W. (1969) J. Mol. Biol. 43,461-477. 26. Traub, W., Shmueli, U., Suwalsky, M. & Yonath, A. (1967) in Conformation of Bio-

27. Inouye, K., Sakakibara, S. & Prockop, D. J. (1976) Biochim. Biophys. Acta 420,

28. Kobayashi, Y., Sakai, R., Kakiuchi, K. & Isemura, T. (1970) Biopolymers 9, 415-

29. Sutoh, K. & Noda, H. (1974) Biopolymers 13,2461-2474. 30. Schwarz, M. & Poland, D. (1974) Biopolymers 13,687-701. 31. Kuntz, I. D., Jr . & Kauzmann, W. (1974) Aduan. Protein Chem. 28,239-345. 32. Berg, R. A,, Kishida, Y., Kobayashi, Y. K., Inouye, K., Tonelli, A. E., Sakakibara, S.

33. Bansal, M., Ramakrishnan, C. & Ramachandran, G. N. (1975) Biopolymers 14,

34. Berg, R. A., Olsen, B. R. & Prockop, D. J. (1970) J. Biol. Chem. 245,5759-5763. 35. Bruckner, P. (1975) Ph.D. thesis, University of Basel, Basel, Switzerland.

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Received April 28, 1976 Accepted July 20,1976

the triple helix ⇌ coil conversion of collagen-like polytripeptides in aqueous and nonaqueous...

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