J. Phys. Chem. 1995, 99, 424-430
424
Calorimetric Investigation of the Coil-Helix Transition of Protamine Induced by Dehydration in Heterogeneous Solutions. Interactions between Double-Stranded DNA and Salmine with Helical Form Akihiro Kagemoto,* Nozomu Fujita, Ken-ichi Ueno, and Yoshihiro Baba Laboratory of Chemistry, Department of General Education, Osaka Institute of Technology, Asahi-ku, Osaka 535, Japan Received: March 9, 1994; In Final Form: August 12, 1994@
The mechanism of complex formation between protamine and a double-stranded DNA (dsDNA) is studied by means of microcalorimetry and spectropolarimetry. From the calorimetric and spectral results, a possible driving force leading to complex formation based on the interaction of the dsDNA with salmine is dominated by cooperative actions between two factors: the first is the electrostatic interaction between the PO4- group in the main chain of the dsDNA and the C(NH2)2+ group of the arginine residue in salmine, and the second is the effect of dehydration accompanying the dsDNA-salmine complex formation induced by the reverse transition from a coiled conformation to a helical one of salmine. To obtain information concerning the conformational change of salmine accompanying the dehydration by interacting with the dsDNA, DSC measurements of salmine solutions with various concentrations of 2,2,2-trifluoroethanol added to salmine with a coiled conformation in aqueous solutions are carried out. The change in enthalpy, AHHc-+, for the reverse transition from a coiled conformation to a helical one of salmine is estimated to be about 2.1 kJ/(mol arginine residue). From the enthalpy cycle, the enthalpy change, A&ehy, of the dehydration accompanying the interaction is also estimated to be about 6.7 kJ. We suggest that when the salmine molecule having a random coiled conformation binds to the dsDNA, the salmine molecule transforms into the a-helical form and is able to bind along the groove of the dsDNA and its enthalpy change is estimated to be about - 11.O kJ/(mol arginine) as the electrostatic interaction involves the C(NH2)2+ group of the arginine residue with a positive charge in salmine and the PO4- group with a negative charge in the main chain of the dsDNA, taking into consideration M c - h and @dehy values.
1. Introduction It is well-known that the DNA molecule in the cell nuclei of an eucaryote exists as chromatin with a higher-order structure which is made up by an interaction with the basic nuclear protein called Furthermore, in the course of fish spermatogenesis, histone within chromatin is usually replaced by another basic nuclear protein called p r ~ t a m i n e ~which - ~ is a specific protein for sperm nuclei of fish. The main reason for the replacement of histone with protamine is the formation of a more compact complex with the DNAs to completely inhibit the transcription." That is, the function of protamine is to protect the DNAs by making up the higher-order structure based on an interaction of DNA with protamine. As concems an interaction mode between DNA and protamine, an electrostatic interaction between the Po4- group with a negative charge in the DNA backbone and the C(NH2)2+ group with a positive charge in the arginine residue in protamine is expected. Several models of nucleoprotamine have been proposed in which the protamine molecule is located in the major or minor grooves of a double-stranded DNA based on the results of X-ray On the other hand, in the crystal structure of protamine, from the single crystal X-ray diffraction studies of protamine binding to transfer RNA (tRNA), it was reported that the protamine molecule had transformed into a conformation with a-helices from a random coiled conformation accompanying the binding to tRNA and that the a-helical conformation
* To whom correspondence should be addressed. @
Abstract published in Advance ACS Abstracts, November 15, 1994.
0022-365419512099-0424$09.00/0
forming a segmented cylinder binds approximately along a minor groove of a double-helical portion of tRNA.14 No information, however, has been established for the molecular interpretation concerning an interaction of a-helical protamine with a double-stranded DNA in aqueous solution. It is considered that information about the thermodynamic quantities accompanying complex formation and the stability of a complex has given us essential physicochemical properties concerning the mechanisms of interaction and the states of hydration and/or dehydration. Particularly, the role of the hydration and/or dehydration accompanying the interaction seems to be an unavoidable problem to elucidate the mechanism of complex formation. In this paper, in order to clarify the molecular interpretation for the mechanism of complex formation between a doublestranded DNA and a-helical protamine, an interaction between a double-stranded DNA and salmine has been studied using a microcalorimeter and a spectrophotometer, respectively. We will discuss the mechanism of an interaction between a double-stranded DNA and salmine with an a-helical conformation by taking into consideration the effects of the hydration and/or dehydration surrounding a double-stranded DNA and/ or salmine.
2. Experimental Section
Materials. The double-stranded DNA (dsDNA) sample used in this study was salmon testes DNA which was purchased from Sigma Chemical Co. The dsDNA solution containing 2 x lo-' mol dm-3 NaCl was sonicated under the experimental conditions of a sonic with 20 kHz and 60 W using a sonicator (UD201, 1995 American Chemical Society
J. Phys. Chem., Vol. 99, No. I , 1995 425
Coil-Helix Transition of Protamine Tomy, Japan) equipped with a microtip according to a usual method.15 Furthermore, in order to remove the contaminants such as proteins contained in the sonicated dsDNA, the sonicated dsDNA solution was purified according to a usual method such as phenol-chloroform extraction,16 and the dsDNA was precipitated with ethanol and CH3COONa.l6 The sonicated and purified dsDNA was dissolved into a 1.0 x mol dm-3 sodium phosphate buffer solution at pH 7.0. The molecular weight of this sonicated dsDNA was about 2 x IO5 (300 base pairs) with a double-stranded helical structure determined from the results of an analysis according to the retention time of high performance liquid chromatography with a Genpak-DNA ion exchange column (Waters, USA) and also from agarose gel electrophoresis. The dsDNA concentration was determined spectrophotometricallyusing the molar extinction coefficient at 260 nm, €260 = 6400 cm-'/(mol nucleotide)." A single-stranded DNA (ssDNA) was prepared by rapid cooling in an ice bath after being kept for 15 min in a water bath controlled at 368 K. To elucidate whether or not a singlestranded structure was obtained, the temperature dependence of an absorbance at 260 nm of this solution was measured by a spectrophotometer. From the results, it was c o n f i i e d that the absorbance at 260 nm for this heat-denatured DNA does not change appreciably in the region 300-370 K. The protamine sample used in this study was salmine18 as free base extracted from salmon sperm, which was purchased from Sigma Chemical Co. and used without any purification. Since the pH of salmine dissolved into a 1.O x mol dm-3 sodium phosphate buffer solution was higher (about 12), a small amount of HCl(1 mol was added to this solution to adjust to pH 7.0. The concentration of salmine was determined spectrophotometrically using the molar extinction coefficient at 220 nm, €220 = 720 cm-l/(mol arginine) at pH 7.0. Finally, the water used to prepare all samples in this study was ultrapure (18.3 M a ) , purified using a MILLI-Q-SP reagent water system with a filter of 0.22 p m (Millipore). Apparatus and Procedure. The apparatus used in this study included calorimeters such as a differential scanning calorimeter (DSC; MC-2D, Microcal) and an isothermal titration calorimeter (OMEGA, Microcal). For the DSC measurements, the dsDNA concentration was about 5.0 x mol dm-3, and the salmine solution with a concentration of 2.0 x mol dm-3 was measured in mixed solvents with various amounts of 2,2,2trifluoroethanol. The heating rate used in this study was about 1 K min-'. For the measurements of the heats of ~ x i n g a, salmine solution with a concentration of 2.5 x mol dm-3 was successively titrated into dsDNA andor ssDNA solutions with concentrations of 2.0 x mol dm-3 in the reaction cell, and the solution was stirred at 400 rpm to obtain a homogeneous solution. The reference cell was filled with water. The spectropolarimeter used in this study was J-20A (JASCO) with a 0.1 mm path length cuvette. For CD measurements, salmine solutions at a given concentration of 2.0 x mol dm-3 in 1.0 x mol dm-3 sodium phosphate buffer solutions containing various amounts of 2,2,2-trifluoroethanol (TFE) (pH 7.0) were used. An IR absorption spectrophotometer used for the measurement of the solid state of salmine was a Fourier transform infrared spectrometer (IR8100, Shimadzu, Japan).
3. Results and Discussion 3.1. dsDNA-Salmine Complex. In order to obtain information about the thermal stability of dsDNA in the presence of salmine, the thermal behaviors of solutions with various
d i L
.............................
(C)
j h J L . . . . . .. . .
4
320
,
n
330
,
o
340
'
n
'
350
r
360
'
8
370
Temperature I K
Figure 1. DSC curves for sonicated salmon testes dsDNA (a) and the dsDNA-salmine system for molar ratios, r, of arginine to nucleotide of (b) r = 0.33, (c) r = 0.50, (d) r = 0.83, and (e) r = 1.0 in 1.0 x sodium phosphate buffer (pH 7.0). Scanning rate empolyed is 1 K min-'.
concentrations of salmine at a given concentration of dsDNA were studied by using the differential scanning calorimeter (DSC). Tipical DSC curves are shown in Figure la-e. As seen in Figure la, the DSC curve of sonicated dsDNA in the absence of salmine shows a broad endothermic peak attributed to the helix-coil transition of the dsDNA similar to that of the unsonicated one. The transition temperature, Tt", and the change in enthalpy, AHto,estimated from the endothermic peak top and area of the DSC curve are 343 K and 13 kJ mol-', respectively. This result is in good agreement with that for unsonicated DNA. While, as shown in Figure lb-e, the transition temperature, T,, of the dsDNA in the presence of salmine shifts to a higher temperature in comparison with Tto. With an increase of the salmine concentration, the DSC curves show double endothermic peaks which are attributed to the biphasic transition of DNA in the presence of ~ a l m i n e ' ~ -similar ~l to that for other basic proteins such as p o l y - ~ - l y s i n e poly-L-arginine,22 , ~ ~ ~ ~ ~ ~ ~ ~ and hist0ne.2~-~~ With further increases in the salmine concentration, the DSC curves at lower transition temperatures gradually become a broad endothermic peak and then disappear; at the same time, the DSC curve at a higher transition temperature gradually becomes larger and finally, at r = 1.0, a sharp endothermic peak appears at 363 K. The dependence of the transition temperature, Tt, of the dsDNA on the molar ratio, r, of the arginine residue in salmine to the dsDNA nucleotide is shown in Figure 2. As seen in a Figure 2a, the transition temperature, Tt-l, of the DSC curve at a lower temperature shifts to a higher temperature with increasing r, and that, Tt-z, at a higher temperature is nearly independent of r. With further increasing r, Tt-l disappears completely at r = 1.0; however, a sharp endothermic peak at 363 K remains, demonstrating that the dsDNA-salmine complex is formed by dsDNA interacting with salmine. We suggest that the 1:l complex between the arginine residue in salmine and the dsDNA nucleotide is formed. On the other hand, the dependence of the change in enthalpy, AHt, estimated from the total areas of the double endothermic peaks on r is shown in Figure 2b. As seen in Figure 2b, AHt increases gradually and is an asymptote at about 15.2 kJ/(mol nucleotide) at r > 0.25. Under the assumption that AHt is the sum of the change in enthalpy, AHt", of the helix-coil transition of the dsDNA at r = 0 and that, AH&ss,for the dissociation of salmine from the
Kagemoto et al.
426 J. Phys. Chem., Vol. 99, No. I, 1995
r:[orgininel/[nucleotidel 0
330
0.5
1.0
1.5 2.0 2.5
1 L
.E
-0.5
m
g z
-1.0
d
5
F 0
0.2 0.4 0.6 0.8 1.0 r:[argininel/~nucleotidel
is moles of dsDNA nucleotide. Tt-l (0)and Tt-2 (A) are the transition temperatures at lower and higher endothermic peaks of the DSC curves.
TABLE 1: Heats of Mixing, A-Hl, for the dsDNA-Salmine System and those, A&T{, Converted per Mole of Arginine for Various Molar Ratios, r, of A r p Residue in Salmine to DNA Nucleotide in 1.0 x 10- mol dm-3 Sodium Phosphate Buffer Solution (pH 7.0) at 310 K
-0.17 -0.33 -0.49 -0.65 -0.82 -0.99 -1.18 -1.35 -1.54 -1.75 -1.95 -2.05 -1.96
-2.13 -2.09 -2.09 -2.06 -2.07 -2.08 -2.10 -2.11 -2.13 -2.16 -2.19 -2.10 - 1.84
1.15 1.24 1.33 1.42 1.50 1.59 1.69 1.78 1.87 1.96 2.06 2.15
-1.84 -1.76 -1.70 -1.67 -1.65 -1.63 -1.61 -1.59 -1.58 -1.57 -1.57 -1.56
d"
-2.5
Figure 2. Plots of the transition temperature, Tt, and the change in enthalpy, AHt, for the dsDNA-salmine complex against r. mol here
0.08 0.16 0.24 0.32 0.40 0.48 0.56 0.64 0.73 0.81 0.89 0.98 1.06
-1.5
-1.60 - 1.42 -1.29 -1.18 -1.10 -1.02 -0.95 -0.90 -0.85 -0.80 -0.76 -0.73
r denotes the molar ratio of arginine residue in salmine to DNA nucleotide. mol is moles of the dsDNA nucleotide. mol is moles of arginine residue in salmine. (I
dsDNA-salmine complex, AHdjss can be determined by subtracting AHi' from AHi such as A H d i s s = AHi - AH?. Since AHt = 15.2 kJ mol-' as an average value at 0.25 < r < 1.0 and AH; = 13.0 kT mol-' at r = 0, then can be estimated to be about 2.2 kJ mol-' (mol here is a moles of nucleotide). 3.2. Heats of Mixing for dsDNA with Salmine. In order to obtain further information on the change in enthalpy resulting from an interaction between the dsDNA and salmine, the heats of mixing of the dsDNA and salmine solutions were measured at 310 K using an isothermal titration calorimeter. The heats of mixing proved to be exothermic, demonstrating that the dsDNA-salmine complex forms. The heats of mixing, AmixHl converted per mole of nucleotide are summarized in Table 1. The results obtained are shown in Figure 3a, where A-Hl is plotted against the molar ratio, r, of arginine to dsDNA nucleotide. As seen in Figure 3a, the absolute value of A-Hl increases by r = 1.0, demonstrating that the stoichiometrically 1:1 complex between the arginine
Figure 3. Plots of the heats of mixing (a) AmH1 per mole of nucleotide and (b) A,,,&' per mole of arginine in salmine against the molar ratio, r, of the arginine residue in salmine to the DNA nucleotide for the dsDNA-salmine system in 1.0 x mol dm-3 sodium phospahte buffer (pH 7.0) at 310 K.
residue and the dsDNA nucleotide is formed; however, the other amino acid residues such as proline, serine, valine, and glycine cannot interact directly with dsDNA. This is consistent with the results of the DSC study described in the preceding section and the results of light scattering intensity measurement^.^' With further increasing r, AmixH1 at r > 1.0 shifts slightly in an endothermic direction and reaches a definite value, indicating that the effect of dehydration accompanying the interaction of salmine with dsDNA is larger. A-Hl for each r is converted to the change in enthalpy A-HI'/(mol arginine residue). The results obtained are listed in the last column of Table 1 and shown in Figure 3b, where A-Hl' is plotted against r. As seen in Figure 3b, A-Hl' shows a definite value of about -2.2 kT/(mol arginine residue) by r < 1.O. This is in good agreement with that for the reverse sign of AHdjssestimated by DSC. It is suggested that the change in enthalpy, AH1, for the formation of the dsDNA-salmine complex is about -2.2 k J mol-' from the results of the calorimetric measurements. 3.3. Dependence on Ionic Strength. In order to obtain information about the dependence of the dsDNA-salmine complex on ionic strength, the heats of mixing of the dsDNA and salmine solutions with various NaCl concentrations were also measured by using the same calorimeter. The heats of mixing for all systems proved to be exothermic and are shown in Figure 4a, where the heat of mixing, A-H2, is plotted against r. Figure 4b also shows the plots of the change in enthalpy, A k H i , converted per mole of arginine residue for all systems with various Na+ ion concentrations against r. As seen in Figure 4b, the absolute value of A-H2' for each ionic strength decreases and that transformed to the endothermic direction at r > 1.O gradually becomes smaller with increasing ionic strength of the Na+ ion. It is worth noting that the stoichiometric ratio at r = 1.0 without Na+ ions shifts to higher r and finally approaches zero with an increase of the ionic strength of the Na+ ion. However, it is very difficult to exactly analyze the behaviors of AmH2' at r 7 0.1 for each system due to a lack of some information. The definite values of A-Hi for each system at r < 1.0 correspond to the change in net enthalpy, A H 2 , of dsDNA-
J. Phys. Chem., Vol. 99, No. 1, 1995 427
Coil-Helix Transition of Protamine 0
2
-0.5
-1.0 o
1 '$ L-
01
! b
-1.0
2
-1.5
4
TABLE 2: Heats of Mixing, A*&, for the ssDNA-Salmine System and those, A&3', Converted per Mole of Arginine for Various Molar Ratios, r, of the Arginine Residue in Salmine to DNA Nucleotide in 1.0 x mol dm-3 Sodium Phosphate Buffer Solution (pH 7.0) at 310 K
r:[arginine]/[nucleotide] 1.0 2.0 3.0
1
"
"
'
'
'
f
0.08 0.16 0.24 0.32 0.40 0.48 0.56 0.64 0.73 0.81 0.89 0.98 1.06
-11.1 -11.4 -11.3 -11.2 -11.2 -11.1 -11.0 -10.9 -10.7 - 10.5 -10.3 -9.99 -9.24
-0.87 -1.78 -2.67 -3.56 -4.43 -5.30 -6.15 -6.99 -7.79 -8.53 -9.23 -9.78 -9.83
1.15 1.24 1.33 1.42 1.50 1.59 1.69 1.78 1.87 1.96 2.06 2.15
-9.75 -9.65 -9.59 -9.52 -9.48 -9.46 -9.44 -9.45 -9.46 -9.48 -9.50 -9.54
-8.46 -7.79 -7.23 -6.73 -6.30 -5.93 -5.60 -5.32 -5.06 -4.83 -4.62 -4.43
r denotes the molar ratio of arginine in salmine to DNA nucleotide. mol is moles of DNA nucleotide. mol is mole of arginine residue in U
-2.5 L
Figure 4. Plots of the heats of mixing (a) A&Hz per mole of nucleotide and (b) A-H; per mole of arginine in salmine against the molar ratio, r, of the arginine residue in salmine to the DNA nucleotide for the dsDNA-salmine systems containing 0.01 (0),0.16 (O),0.31 (A), and 0.51 (W) mol dm-3 Na+ ions (pH 7.0) at 310 K. salmine complex formation and its values are estimated to be about -0.9 kJ mol-' for 1.6 x lo-' mol dm-3 Na+ ion, -0.34 kJ mol-' for 3.1 x lo-' mol dm-3 Na+ ion, and -0.17 kJ mol-' for 5.1 x lo-' mol dm-3 Na+ ion. From these results, it is considered that dsDNA-salmine complex formation is inhibited by an electrostatic interaction between the P04- group in the dsDNA and the Na+ ion. In other words, the driving force leading to dsDNA-salmine complex formation is dominated by the electrostatic interaction between the PO4- group in the dsDNA and the C(NH&+ group of the arginine residue in the salmine molecule. 3.4. Effect of Dehydration. As one approach to obtain information about the reason why the change in enthalpy of the dsDNA-salmine complex formation induced by the electrostatic interaction demonstrated from the calorimetric measurements is very small, the effects of the hydration and/or dehydration surrounding the dsDNA and/or salmine accompanying the interaction are also studied from the heats of mixing of a single-stranded helical structure of the DNA (ssDNA) and salmine. The heats of mixing of the ssDNA-salmine system proved to be exothermic, indicating that an interaction between ssDNA and salmine exists. The heats of mixing, Ami~H3, converted per mole of nucleotide are listed in Table 2 and shown in Figure 5a, together with AmiTH1 for the dsDNA-salmine system, where AfixH3 is plotted against r. As seen in Figure 5a, A d H 3 shows the same behavior as A k H 1 , except that the magnitude of A-H3 as an absolute value is larger than that of A-Hl, A-H3 for each r is converted to the change in enthalpy, A&H3/ per mole of arginine residue. The results are summarized in the last column of Table 2, and A-H{ is plotted against r as shown in Figure 5b, together with A,,&'' of the dsDNA system. The change in net enthalpy, A H 3 , for the interaction between the ssDNA and salmine is estimated to be about -11.O kJ mol-' by extrapolation to r = 0. The absolute value of A H 3 for the ssDNA-salmine system is larger than that of AH1 for the dsDNA-salmine one. Assuming that the interaction mode between the ssDNA and salmine is the same as that of the dsDNA-salmine system, the
salmine. r:[orgininel/[nucleotidel 0
0.5
1.0
1.5 2.0
2.5
-11 1
a
-13
Figure 5. Plots of the heats of mixing (a) A-H3 per mole of nucleotide and (b) A - H i per mol of arginine in salmine against the molar ratio, r, of the arginine residue in salmine to the DNA nucleotide for the ssDNA-salmine system (A), together with the dsDNA-salmine system (0)in 1.0 x lo-* mol dm-3 sodium phosphate buffer (pH 7.0) at 310 K.
difference, AAH, between A H 3 and AH1 seems to correspond to the change in enthalpy of contribution of dehydration surrounding the dsDNA and/or the salmine molecules which is brought about by the interaction of the dsDNA and/or the ssDNA with salmine. As concerns the effects of water molecules interacting with the dsDNA, it has been that the double-stranded helical structure depends on the number of water molecules hydrated to the dsDNA, and Herskovits et aL3' also suggested that the structure of the dsDNA interacting with protamine inhibits the normally occurring conformational change of the dsDNA and is stabilizing the B-form against dehydration based on spectroscopic studies. The reason that the absolute value of AH1 is smaller than that of A H 3 is the contribution of the great dehydration that occurs only when salmine binds to the dsDNA rather than the
Kagemoto et al.
428 J. Phys. Chem., Vol. 99, No. I, 1995 4 ,
I
-
L
C
I
I
I
\
Q' -12 -16 I 200
220
240
u
I
260
I
A/nm
-----v-
293
W
313
333
353
I
373
T/K Figure 7. DSC curves for the conformational change of salmine in mixed solvents with various volume percents of TFE in water: (1) 0/100, (2) 10/90, (3) 20/80, (4) 30/70, and ( 5 ) 50/50 TFWwater.
I
01 2000
I
1646 I
1800
I
1600 1400 W o v enu m b er / c m''
4 1200
1000
Figure 6. (a) CD spectra of salmine dissolved in (1) 1.0 x mol dm-3 sodium phosphate buffer at pH 7.0 and (2) 30/70 (v/v), (3) 50/ 50 (v/v),and (4) 70/30 (v/v) ( ~ 0 1 %TFE (2,2,2-trifluoroethanol)/water mixed solvents (pH 7.0) at 298 K. (b) IR spectrum of the solid state of salmine at 298 K.
ssDNA. Assuming that the reason for the great dehydration in the formation process of the dsDNA-salmine complex is the conformational change of the salmine molecule, it can be expected that the salmine molecule is able to transform into an a-helical conformation from a random coiled one induced by the dehydration accompanying the formation of the dsDNAsalmine complex. 3.5. Coil-Helix Transition of Salmine Induced by Dehydration. As an approach to confirm the effects of dehydration on the conformational change of the salmine molecule, CD spectra of the salmine molecule in mixed solvents with 1.0 x mol dm-3 sodium phosphate buffer solutions containing various amounts of 2,2,2-trifluoroethanol (TFE) were measured at room temperature. The results are shown in Figure 6a. As seen in Figure 6a, the salmine molecule in aqueous solution shows a CD spectrum characteristic of the random coiled confomation. However, the salmine solution with a TFE concentration of 30 vol % (% (v/v)) in mixed solvents shows a CD spectrum with negative maxima at wavelengths of 208 and 222 nm, showing the characteristic helical conformation, demonstrating that the conformation of salmine transforms into a helical conformation from a random coiled one with increasing the TFE concentration in mixed solvents. Recently, Storrs et aL3* studied the effect of TFE on the helical stability of peptides and provided evidence that this stabilizing effect is a general solvation effect and not a specific interaction of the helical peptide with TFE; furthermore, TFE unstabilizes the coiled state by less effective hydrogen bonding of the peptide amide. From this viewpoint, it is expected that the TFE molecule breaks down the hydrogen bonds between the water molecule
and peptide amide of salmine and the salmine molecule forms an a-helical structure based on the intramolecular hydrogen bond in salmine. Hence, from the results of CD spectra, it is suggested that the dehydration is the driving force that induces the conformational change from a random coiled conformation to a helical one in aqueous solution. Furthermore, the conformation of salmine in the solid state is studied by Fourier transform infrared spectrophotometry (FT-IR) at 298 K. The IR spectrum obtained is shown in Figure 6b. From the observed frequencies of the amido-I and -11bands at 1646 and 1543 cm-', it is demonstrated that these band assignments have been made for an a-helical conformation. From the results of CD spectral measurements in mixed solvents and IR spectral measurements in the solid state of salmine, we suggest that the dehydration from salmine induces the conformational change of salmine to a helical conformation from a random coiled one. These results seem to support the calorimetric ones obtained in the present work. The DSC measurements for salmine in mixed solvents with various amounts of 2,2,2-trifluoroethanol (TFE) were carried out. The DSC curves show an exothermicity as shown in Figure 7. As seen in Figure 7, the DSC curve of salmine in aqueous solution does not show the thermogram, demonstrating that the salmine molecule does not cause in its conformation; in other words, the salmine molecule in aqueous solution exists as a random coiled conformation. However, with increasing TFE concentration in mixed solvents, the DSC curves with an exothermic peak at 328-350 K appear, demonstrating that these exothermic peaks of the DSC curves correspond to the phase transition from a helical conformation to a coiled one of the salmine molecule. Under the assumption that the areas of DSC curves correspond to the change in enthalpy based on the helix-coil transition, the change in enthalpy, M o b s , observed from each DSC curve with various components of TFE in mixed solvents can be determined. The plot of m o b s against the TF% concentration is shown in Figure 8, here M o b s is plotted as the change in enthalpy (endothermic) based on the coil-helix transition. As seen in Figure 8, f?&I,s increases with increasing TFE concentration and then is an asymptote at AH& = 44.0 kJ/(mol salmine) at the concentration 100 vol % TFE. The composition transition of salmine occurs sharply in the neighborhood of 30 vol % TFE in mixed solvents. While the difference, A[@],between the molar ellipticity [e]for the salmine molecule at a TFE concentration of zero and that for each component of the TFE in the mixed solvent is estimated from the CD spectra of Figure 6a, and the results are also shown.in
Coil-Helix Transition of Protamine
J. Phys. Chem., Vol. 99, No. I, 1995 429
5 0 7
I ,+' ,
.'
I
0",4
,
,
0
,
, 50
Since M c - h = 8.8 kT and M c - h " = 2.1 kJ, estimated in the preceding section, then A H d e h y is estimated to be about 6.7 kJ. It is suggested that this value corresponds to the change in enthalpy based on the dehydration of the salmine molecule accompanying the dsDNA-salmine complex formation. From the results obtained above, the net enthalpy change, A H b i n d , for the binding of the dsDNA with salmine can be presented as
1, 0.5
,
,
,
2,
(5)
,
100
Concentration o f TFE
(%)
Figure 8. Plots of
A H o b s - l (0) and A[O] (0) accompanying the conformational change of salmine in mixed solvents against the concentration of TFJ3.
Figure 8. The behavior of A[e] is similar to that for calorimetric results, suggesting that the salmine molecule transforms into the a-helical conformation from a random coiled one with an increase of the TFE concentration and its change in enthalpy, can be estimated to be about 2.1 kJ converted per mole of arginine residue. 3.6. Interaction between dsDNA and Salmine with Helical Form. The change in enthalpy of the interaction between the dsDNA and salmine can be analyzed according to the following enthalpy cycle ssDNA
* dsDNA
+
salmine (coil) AHg
ssDNA-salmine (coil)
salmine (coil)
AHt
]
AH1
dsDNA-salmine
(helix)
AH;
AHe-h
where AHf is the change in enthalpy accompanying the transformation to the dsDNA-salmine (helix) complex from the ssDNA-salmine (coil) one, AH1 and A H 3 are the changes in enthalpy of the complex formations between the dsDNA and salmine and between the ssDNA and salmine, AH? corresponds to the formation of the double-stranded helical structure of DNA with the reverse sign of the change in enthalpy, AHt" for the helix-coil transition of the dsDNA is obtained from DSC. From the enthalpy cycle
AH,=AHH,0'+AH1-AH3
(2)
Since AH,"'= - 13.0 kT, AH1 = -2.2 kJ, and A H 3 = -11.0 kJ, then AHf is estimated to be about -4.2 kJ/(mol arginine residue). However, AHf in eq 2 is the sum of Milto'and m c - h for the formation of a helical structure of salmine from a coiled conformation accompanying the interaction with the dsDNA as follows
AHf = AH;'
Since AH1 = -2.2 kJ from the enthalpy cycle and AHc+ = 8.8 kJ, then A H b m d is estimated to be about -11.0 kJ/(mol arginine residue), suggesting that this M b h d value corresponds to the change in enthalpy based on the binding between the dsDNA and salmine with an a-helical conformation. It is concluded that since the dsDNA with its steric structure containing minor and/or major grooves existing in a double helical conformation has more binding affinity compared with ssDNA without grooves, the salmine molecule, transformed into an a-helical conformation, binds along the grooves of the dsDNA, and its binding mode is an electrostatic contribution between the PO4- group with a negative charge of the main chain of dsDNA and the C(NHz)z+ group with a positive charge of the arginine residue in salmine.
4. Conclusion
AH;'
+
1
(4)
+ AHc-h
(3)
Since AHf is -4.2 kJ from eq 2, and AHt"' is -13.0 kJ, then M c - h is estimated to be 8.8 kJ/(mol arginine). But this AHc+ value is considered the sum of the change in net enthalpy, M c - h ' and the enthalpy change, M d e h y , of the dehydration accompanying the conformational change of salmine by interacting with dsDNA as
The mechanism of complex formation between dsDNA and salmine has been studied by means of microcalorimetry and spectropolarimetry. From these results, the driving force in making up the dsDNA-salmine complex is the cooperative actions between two factors, namely, the electrostatic interaction between the PO4- group with a negative charge of the main chain of dsDNA and the C(NH&+ group with a positive charge of the arginine residue in salmine and the conformation of salmine being transformed into a helical conformation from a random coiled one as the reverse transition induced by the dehydration acompanying the interaction taking into consideration the results concerning the conformation of the salmine molecule from the dependence of the TFE concentration in mixed solvents obtained by spectral and the calorimetric measurements. It is concluded that, by taking into consideration of the effects of the dehydration and conformational change of salmine, the change in net enthalpy, A H b i n d , of binding based on the electrostatic interaction between the dsDNA and salmine with an a-helical conformation is determined to be - 11.O kJ/(mol arginine residue) from the enthalpy cycle.
References and Notes (1) Strver. L. Biochemistrv. 3rd ed.: W. H. Freeman and ComDanv: I . New. York,'1988. (2) Alberts, B.: Brav, D.; Lewis, J.: Raff, M.: Roberts, K.; Watson, J. D. Molecular Biology ojthe Cell, 2nd ed.; Garland Publishing, Inc.: New York, 1989. (3) Kornberg, R. D.;Mug, A. Sci. Am. 1981,244, 48. (4) Watson, J. D.; Hopkins, N. H.; Roberts, J. W.; Steitz, J. A.; Weiner, A. M. The Molecular Biology of the Gene, 4th ed.;The Benjamin/Cummings Publishing Co., Inc.: New York, 1987. (5) Ando, T.;Yamasaki, M.; Suzuki, K. Proramines-Characterization, Structure and Function; Springer-Verlag: Berlin, 1973. (6) Hecht, N. B. Mammalian protamines and their expression. In Histones and Other Basic Nuclear Proteins; Hnlica, L. S . , Stein, G. S., Stein, J. L., Eds.; CRC Press: Boca Raton, 1989. (7) Willkins, M.H. F.; Randall, J. T. Biochim. Biophys. Acta 1953, 10,192.
