VOL. 15, 687-706 (1976)
BIOPOLYMERS
Structural Investigations on DNAeProtamine Complexes T. T. HERSKOVITS* and J. BRAHMS, Institut de Biologie MolBculaire (C N.R.S.), Uniuersite de Paris V I I , Paris 5", France
Synopsis Protamine-DNA complexes in film and in solution have been investigated by means of infrared linear dichroism, ultraviolet circular dichroism, and laser Raman spectroscopy. At high relative humidity and in solution both infrared linear dichroism and ultraviolet circular dichroism indicate the presence of a modified B form of DNA (designated as B* in our other paper^^^.^^,^^). This modified B form is characterized by a change of the orienA
tation of the OPO bisector angle by about 4O with respect to the helical axis when compared to the B form of DNA. At decreasing relative humidities the same modified B form is maintained and no structural transitions B A (or B C) normally occurring in free DNA were observed. The absence of the A form in these complexes was also confirmed by laser Raman scattering studies of protamine-DNA complexes. On the basis of these results, a model of the protarnineDNA complex is proposed, which suggests that the presence of apolar amino-acid residues, and probably the folding of the polypeptide chain, is responsible for preventing the B-to-A transition; this occurs either by protecting the highhumidity modified B form against dehydration or by steric interference of this protein probably located in one of the DNA grooves.
-
-
INTRODUCTION The nucleoprotamines represent one of the simpler forms of DNAbasic-protein complexes found in eukaryotic cells. The study and characterization of the complexes formed between DNA and the protamines found in sperm nuclei of some fish and mollusks are subject to fewer difficulties of interpretation of physical data associated with the study of histone and basic synthetic polypeptide complexes of DNA and of chromatin.1-8 On the basis of X-ray diffraction studies of nucleoprotamines of sperm heads and DNA-protamine complexes, Wilkins and co-worke r ~ , ~and , ~more , ~ ~recently Subirana and Puigjaner,'l have suggested that the DNA in the nucleoprotamine complex assumes a B structure with the protamines filling the small groove of the DNA double helix. X-ray scattering studies on gels3 and early polarized infrared studies of Bradbury et a1.I2 on oriented films are largely consistent with these basic features of the organization of the nucleoprotamine particle. However, due to a less expressed orientation, the structural parameters * On leave from the Department of Chemistry, Fordham University, Bronx, N.Y. 10458. 687 0 1976 by John Wiley & Sons, Inc.
688
HERSKOVITS AND BRAHMS
of DNA and of proteins in complexes are relatively poorly known. A current method that can yield precise structural parameters of the orientation of different groups in different conformations of DNA is infrared linear d i c h r o i ~ m . ' ~ - ~ ~ One approach to the study of the conformation of DNA and proteins in nucleohistones and nucleoprotamines related to the structural organization of chromatin is to study the properties of reconstituted DNAprotein complexes and the complexes of DNA with synthetic polypepProtamines are a family of very similar proteins and they are the main constituents of DNA complexes in spermatozoa of fish. They have been named on the basis of their origin (e.g., clupeine from herring, protamine is also used to designate this protein from salmon sperm). This paper reports the results of the initial phase of our studies of the simpler DNA-protamine complexes carried out simultaneously on both oriented films and on solutions prepared from the same complexes. In addition to the results obtained by infrared dichroism methods we also report the results of circular dichroism (CD), Raman scattering, and thermal denaturation measurements.
MATERIALS A N D METHODS Materials Protamine chloride (Grade V), clupeine sulfate, and calf thymus DNA were purchased from Sigma Chemical Company. Salmon sperm DNA was obtained from Worthington; protamine sulfate, from Fluka. Spectra-pore 3 dialysis tubing, with a molecular-weight cutoff limit of 3500, purchased from Spectrum Medical Instruments (Los Angeles, Calif.), was used for most of our studies. Some of the earlier experiments including the work with 2:l complexes of protamine and DNA preparations were carried out using dialysis tubing that was pretreated using the method of Callanan et a1.18 by overnight drying a t 80OC. DNAaprotamine and DNA-clupeine complexes were prepared according to the salt gradient method of Huang et al.19 Usually 40 mg of DNA dissolved in 1.0 ml of 4.0 M NaCl, pH 7.5 was mixed with 1.0 ml of protamine or clupeine dissolved in 2.0 M NaC1, pH 7. After thorough mixing and standing for several hours or overnight, the initially turbid solutions would clear, a t which stage dialysis would be initiated. The dialysis steps used were 4 hr each against 1.5 M and 0.4 M NaCl, followed by 1hr or more against 0.3 M and 0.15 M NaCl, in the cold. In order to minimize the loss of protein during the initial stages of complex formation, calculated amounts of protamine or clupeine were also added to the dialysates (15 ml each adjusted to pH 7) so as to keep the concentrations constant both inside and outside the dialysis sack. Deuterium oxide solutions were similarly prepared, using approximately 5-ml changes of dialysate of the desired final salt content and adjusted
DNA-PROTAMINE COMPLEXES
689
to approximately 7.5 pH. (We were unable to prepare complexes of salmon sperm DNA by this method, since the initial turbidity of the DNA-protamine mixture would not clear sufficiently to any acceptable level, even after several days of standing a t room temperature.) The stock solutions of several calf thymus DNA complexes, prepared by this procedure, contained approximately 1.5-2% DNA, usually required for the preparation of oriented films and laser Raman studies. For circular dichroism (CD) and thermal denaturation T , measurements, the stock solutions were diluted by a factor 200-300 with 0.05 or 0.15 M NaCl and gentle magnetic stirring in the cold, with final pH of 6.8-7.2. DNA concentration of the complexes was based on absorbance measurements on a Cary 17 recording instrument, using the extinction coefficient of 6500 M-’ cm-I at 260 nm. Methods
Infrared Dichroism. Infrared dichroism measurements were made on a Beckman IR 9 spectrophotometer equipped with a wire grid polarizer in constant humidity cells used in this laboratory.13-15 The humidity of the oriented samples, deposited on “Irtran” plates, was controlled by use of various saturated salt solutions placed in the bottom compartment of the assembled cell in tightly fitted removable Teflon walls.13-15 The dichroic ratio R ( l / l l ) , which is equal to the absorbance ratios A ( 1) to A ( 11) corrected for water and baseline contribution at any of the bands examined, was computed from the spectra of the films recorded with the electrical vector of the polarized light first set perpendicular and then parallel to the orientation axis.I3-l5 This was accomplished by subtracting from the observed absorption a t 1090, 1230, and 1710 cm-l the contribution due to water of the film measured a t 3400 A cm-l.12J3,15 The orientation angles 8 of the OPO bisector and the 0-0 lines of the DNA phosphate groups in relation to the fiber axis were calculated using the dichroic ratios R ( 1 /11) and t h e Fraser relationship: R(1III) = (sin2 8
+ g)/(2 COG6, + g )
(1)
where the parameter g is related to the fraction f of oriented DNA, in the film, with:
f
= 1/(1
+ 3/2g)
(2)
The g and f parameters can be readily evaluated for the B or C form of DNA. Since the bases are oriented nearly perpendicular to the fiber axis of DNA in these two forms, the dichroic ratio at 1710 cm-l produced by the orientation of the bases with 8 taken as 90° gives a value for g = 1/(R1710 - 1). Once g is determined, Eq. (1) or (3) may be used to calculate the two angles associated with the dichroism of the phosphate groups a t about 1090 and 1230 cm-l:
690
HERSKOVITS AND BRAHMS
For the A form with the bases tilted at approximately 20' in relation to the fiber axis of DNA,20the g and f have been evaluated using the approximate angle of 80' for the average transition moments of the base absorption band at 1710 cm-l.15 Raman Spectra. Raman spectra were obtained on a Jarrell-Ash 25400 laser Raman spectrophotometer utilizing a Coherent Radiation model 52 argon ion laser set to 514.5 nm. The temperature of the samples was controlled by means of a brass block sample holder through which water circulated from a constant temperature bath. Capillary tubes held in the horizontal position in the middle of the specially constructed brass block were employed to contain the DNA-protamine and DNAaclupeine complexes. Circular Dichroism. Circular dichroism measurements were performed using a high-sensitivity apparatus constructed in this laboratory. The calibration of the dichrograph was carried out with a solution of 10-camphorsulfonic acid (2.19 mg/ml) taking (AE) = 2.3L21 Thermal Denaturation. Thermal denaturation measurements were made in a Shimadzu QV 50 spectrophotometer on degassed solutions of the various complexes and DNA samples investigated, as previously described.22
RESULTS Figures 1 and 2 show typical ir absorption spectra of oriented DNA. protamine and DNA-clupeine complexes measured a t different relative humidities (rh). In general, one finds all the main features of DNA spectra in the B form with the exception of two regions at 1665 and 1550 cm-l of the amides I and I1 absorption bands of protein. 1) The bands in the region of DNA base absorption 1800-1500 cm-l have been assigned to the C=C, C=N, C=O in-plane ring doublebond stretching vibrations and NH2 deformation vibration^.^^?^^ The band at 1710 cm-l is characteristic of the base-paired struct ~ r e .All ~ these ~ bands are perpendicular with respect to the orientation axis, as one finds in oriented DNA films (Fig. 3c and Refs. 12, 13,15). 2) The presence of bands at about 1230 and 1090 cm-l is assigned to antisymmetric and symmetric phosphate vibrations of DNA.
- -
In DNA the dichroism of these phosphate bands changes drastically when B A or B C transition occurs. The high-humidity B form or B family form is characterized by a predominant perpendicular dichroism of the band at 1090 -l and by the almost nondichroic band at 1230 cm-1.12J3J5 This is clearly observed in the spectrum of the DNAprotamine complex at high rh shown in Figure la. In addition, the B form (and also the C form) is characterized by the presence of an ir band
DNA-PROTAMINE COMPLEXES
1800
1800
1400
1200
H)o
69 1
800
cm-'
Fig. 1. Infrared dichroism spectra of oriented DNAeprotamine complex of 1:1 molar phosphate-to-arginine ratio with different relative humidities. (a) 98%. (b) 76%. (c) 15%. Spectra were obtained with electrical vector of the radiation parallel (- - -) and perpendicular (-) to the orientation or fiber axis of the complex; NaCl content of films 3-5% (w/w).
a t 836 cm-1.26,27 In DNA the transition to the A form at lower humidit y is clearly recognized by a parallel dichroism of the 1090-cm-' band12J5 and by the presence of two bands a t 812 and 860 cm-l shown in Figure 3c (arrows); these bands situated in the region of 750-860 cm-' are undoubtedly characteristic of the DNA backbone phosphodiester chain vibrations in the A or B geometry.27 As shown in Figures 1 and 2 none of the dna-protamine and DNA-clupeine complexes examined at different humidities exhibits the A form. The spectrum of a clupeine film is shown in Figure 3b. The protein contribution is easily observed in the region of amides 1 and I1 absorption bands at about 1660 and 1550 cm-'. The high intensity of these bands is due to a relatively greater amount of protein used for the film formation than for complexes with DNA. In complexes even at relatively lower protamine-to-DNA ratios the protein contribution can be clearly distinguished, particularly in the region of the amide band at about 1500 cm-' a t lower rh (Fig. 3a). We estimate that outside of these regions the protamine contribution to the DNA-protamine cornplexes a t about 1090,1230, and 1710 cm-l will only be 10-15% of the absorbances shown at these frequencies.
692
HERSKOVITS AND BRAHMS
cm-’
Fig. 2. Infrared dichroism spectra of oriented DNA-clupeinecomplex of 1:l molar arginine-to-DNA-phosphate ratio with different relative humidities. (a) 98%. (b) 15%. Spectra were obtained with the electrical vector of the radiation parallel (- -) and perpendicular (-) to the orientation axis of the film; NaCl content of complex 3 4 % (w/w).
-
Thus the DNA characteristic bands such as those of phosphates a t 1090 and at 1230 cm-1 and of paired bases at 1710 cm-l and their orientation with respect to the orientation axis are essentially unperturbed by the contribution of protamines. Further evidence for the conclusion that the orientations at different groups relative to the helical axis are similar in DNA and in DNA-protamine complexes are provided by X-ray diffraction measurements of Wilkins e t aL9J0 and by Subirana and Puigjanerll on fibers of DNA-protamine complexes. Figures 4 and 5 show the plots of dichroic ratios R(III1)of these three bands at 1090, 1230, and 1710 cm-l as a function of relative humidity obtained on some 1:l complexes of DNA-protamine and DNAaclupeine films. This plot of dichroism shows no contribution from the A form in
(a) Fig. 3 (continued)
DNA-PROTAMINE COMPLEXES
693
Fig. 3. Comparison of infrared spectra. (a) DNA-clupeine complex a t 5%rh (the arrow indicates the amide I1 band of protein a t about 1550 cm-'. (b) Clupeine at 15%rh. (c) DNA at 66% rh (the arrows indicate the presence of 812- and 860-cm-l bands characteristic of the A form not observed in the complexes). The spectra of DNA and DNA-clupeine complexes were obtained with the electric vector of the radiation perpendicular (-) and parallel (- - -) to the orientation axis of the film.
the 80-50% rh region, seen with the DNA films by Pilet and Brahms13-15 and almost the absence of structural transitions as a function of relative humidity. In fact Pilet and Brahms13?l5have found that the dichroism of DNA (calf thymus) films (NaC1 content 44% w/w) at 1090-cm-l band changed from perpendicular (e.g. R = 1.3) to parallel values (below unity, R ='0.8); a t the 1230-cm-' band the change in DNA conformation was reflected in an increase in R (from almost 1 to about 1.3). No such
HERSKOVITS AND BRAHMS
694
changes were found in complexes of DNA-protamine and DNALclupeine and only the B-type form was seen at different rh's. This is also confirmed by Subirana and Puigjanerll who have found by X-ray diffraction measurements on fibers of DNA-protamine complexes from three mollusk species that the high-humidity B type of conformation of DNA was also stabilized to changes in relative humidity. This is in contrast with the absence of such an inhibitory effect on structural transitions observed in this laboratory in complexes of DNA with basic polypeptides: poly(L-arginine) or poly(L-lysine).28 Table I presents structural parameters of various 1:l and 2:l complexes (molar ratios of arginine to DNA phosphate) of DNA-protamine and DNA-clupeine films obtained from analysis of the data a t the high (90-100%) rh region. On an average, the orientation OPO bisector angles of the complexes are about 4' lower than the angle of the B form of calf thymus DNA15 (particularly well expressed in 2:l complexes). This is considered as a modified B form. The measured distortion in angular orientation of the phosphate groups of DNA in the complexes in films is consistent with the observed changes in the CD spectra of the complexes in solution in the neighborhood of the 276-nm CD band (Table 11).
r.h.m"c) 47
76
90
95
I
I
I
I
I
DNA-Pmtamine,l:l
2.0
R[l/iI]
5
l.8
-
1.6
-
loo% 1
0
1.2 1.0 1.4
0.8I
a2
I
0.4
I
0.6
4 B
DNA-PROTAMINE COMPLEXES
695
r.h.
2.2
-
5
47
I
1
76869093 I
I
I
I
95
Kn
I
I
DNA-Ckrpeine.l:l
1.8 -
2.0
R[*/l,y
:
1.4
-
1.2
-
1.0
-
n
h
r 3
R1230
0.81
I
I
I
0.4
0.2
1
0.6
I
1.0
0.8
A3400 cm-1 (b) of)the logo-, 1230-, and 1710-cm-' bands Fig. 4. Plots of the dichroism ratios R (1/11 as a function of relative humidity of the films (1:l molar phosphate-to-arginine ratio) described in Figs. 1 and 2. (a) DNA-protamine. (b) DNAdupeine. Films contained in the final state 3-5% NaCl.
Changes in salt concentration of the solutiozls from which the films were cast had only minor effects on the conformationally sensitive 1230-cm-l dichroic band (Fig. 5a and b). It is significant that the dichroic ratios at this band show no obvious change as a function of relative humidity.14 Thus the presence of protamine stabilizes the modified-B-type conformation, designated as B* in our other papers.27728946 TABLE I Structural Parameters of DNA.Protamine and DNAClupeine Complexes at High Relative Humidity (90-100%)
Complex DNA.protarnine 1:l complex DNA.protamine 2:l complex DNAdupeine 1 :1 complex Calf thymus DNA
Film Parameters
Phosphate
Orientation
0(2)0(3) Line
O?O Bisector ( 6 , d (ded
NaCl Content of Film (70)
g
f
1.5-2.5 3-5 7-8 3-5
0.9- 1.2 0.63-0.68 0.71-0.84 1.4-1.7
0.36-0.43 0.42-0.44 0.44-0.49 0.28-0.32
55.0 t 57.0 f 57.6 i 54.0 i
3-5
0.95-1.15
0.36-0.42
53.1 i 0.8
67.9 i 1.0
3-5
2.2
0.23
56
69
( 0 , d (ded 0.4 0.4 0.6
0.3
~_
64.9i 67.5 t 66.9 f 64.9 t
0.9 0.3 0.8 0.6
HERSKOVITS AND BRAHMS
696
Circular Dichroism and Thermal Denaturation Studies The CD spectra of DNA and DNA-potamine and DNA-clupeine obtained by salt gradient dialysis are shown in Figure 6. The spectra show a noticeable reduction in the positive 275-nm band from 2.6 to approximately 2.0-2.2 and a relatively minor change a t the second, negative band near 245 nm. Table I1 presents a summary of the CD data observed a t these two extrema together with the effects of increasing protamine and clupeine in the complexes on the melting temperature T,. In a recent study of the properties of protarnine-DNA complexes, Yu and Li29 have reported very similar changes of the CD spectra of complexes prepared by direct mixing (i.e., decrease of the intensity of the positive band). The results of these solution studies are consistent with the notion that the bound protamine or clupeine alters or distorts the high-humidity B conformation of DNA, as suggested by our film infrared studies described before. Binding of a basic protein like protamine to DNA must occur through phosphate groups of the DNA backbone. Subtle angular readjustment of the phosphate groups is thus expected during the course of complexation with either synthetic or natural polypeptides rich in arginine. Of
r.h. 2 . 2 k 4 m - l l 15324758 l - l *
76 81
DNA-Protamine,l:l
0
2.0 1.8
R" 14 1.2
A3400
90 95 100
cm-1
(a)
Fig. 5 (continued)
%
DNA-PROTAMINE COMPLEXES
697
r.h. 5876 90 95 ‘L 9 15
3.4
32
I
DNA-Protamine,l:l
3.0
0
1.4
I
I
I
0.6
0.4
A3400
I
0.8
I
1.0
cm-’
(b) of the logo-, 1230-, and 1710-cm-’ bands Fig. 5. Plots of the dichroism ratios R (l/il) as a function of relative humidity of 1:l DNA.protamine complexes. (a) At low salt content. (b) At high salt content. The films in the final stage contained 1.5-2.5% and 9-15% NaCl (w/w).
the 30-32 peptide groups in the protamines and clupeine, 20-21 residues are arginine, with many of these groups running in clusters of four or five in the sequence.30 Complex formation of the basic peptide groups also confirms thermal TABLE I1 Circular Dichroism Parameters of DNA (Calf Thymus), DNA.Protamine and DNAClupeine Complexes “276
DNA calf thymus M NaCl, pH 6.9 5x 0.15 M NaCl DNA.protamine 1:1 complex M NaCl, pH 7.0 5x 0.15 M NaCl, pH 6.5 DNA.protamine 1 : 2 complex 5x M NaCl, p H 7.2 D N A d u p e i n e 1:1 complex M NaCl, p H 7.2 5x DNA-clupeine 1:2 complex M NaCI, pH 7.2 5x
“246
Ttn (“C)
2.55 2.58
-3.01 -2.96
65 f 0.5
2.45 2.50
-2.82 -2.85
77,74
2.22
-2.95
73-75
2.22
-2.7
72
2.0
-2.9
76
HERSKOVITS AND BRAHMS
698
f
’\
1.0
-
-2.0-
.
.
DNA-Clupemne
5.W’MNa CI
300
280
260
240
221)
300
1
I
I
280
260
240
I 220
Fig. 6. Circular dichroism spectra of DNA-protamine and DNA-clupeine complexes. (a) DNA-protamine arginine-to-phosphate molar ratio of complex: curve a-DNA alone; curve b-0.6; curve c--1.0; curve d-2.0, in 0.05 M NaCl, pH 7-7.2. (b) DNA-clupeine arginine-to-phosphate molar ratio of complex: curve a-DNA alone; curve b--1.0; curve c-2.0; in 0.05 M NaCl, pH 7.2.
stabilization of the complex. This is shown by the T m data also listed in Table 11. Shifts in the denaturation midpoint T m of the various complexes examined in this paper are roughly proportional to the amount of protamine or clupeine present in the complex. However, unlike the complexes obtained by direct m i ~ i n g , 2 ~ the , ~ l thermal -~~ profiles of the complexes prepared by salt gradient dialysis have revealed no clearly discernible biphasic characte? (results not shown). Differences in the melting behavior of various synthetic and natural polypeptide. DNA complexes have been noted by various authors and may depend on the method of
Raman Scattering Studies Laser Raman spectra of DNA and DNA-protamine and DNA-clupeine complexes obtained a t 25O-28OC on neutral DNA-salt solutions in both water and deuterium oxide solvents are shown in Figures 7 and 8. Figures 8 and 9 also present Raman spectra of deuterium oxide and water solutions of 3% protamine chloride and clupeine sulfate. Since these two sets of solutions had approximately several times the amount of protein present in the various DNA complexes studied, one would not expect clearly discernible contributions to the spectra of the complexes of any of the relatively weak Raman lines outside of the amide band in the region of 1650 cm-l. [The assignment of various Raman active bands in helical and nonhelical polypeptides such as poly(L-lysine) and
699
DNA-PROTAMINE COMPLEXES
TABLE I11 Characteristic Raman Frequencies of Calf Thymus DNA and 1:1 Arginine to Phosphate DNA.Protamine and DNA.Clupeine Complexes in Aqueous and Deuterium Oxide Salt Solutions a t 26" f 2°C (pH-pD 8.0-7.4)a Frequencies (cm-' ) in D , 0
Frequencies ( c m - ' ) in H,O
DNA. Protamine
DNA. Clupeine
DNA Alone
DNA. Protamine
DNA. Clupeine
DNA Alone
788
792
790
791
792
792
830-35
825-30
836-38
836-38
836-38
892
-885
1015 1058 1095
-1015 1059 1094
-1145 -1180 -1186 1230 -1225 1260 1260 1306 1310 1342 1342 1378 1376 1428 1422 -1465 1491 1490 -1515 1535 1581 1582 Broad H,O band 1650-60 cm-' max
-
-
a b
-830-35
898 925 972 1025 1058 1094
- 1005 1058 1095
896 922
895 -920 -965
1057 1093
1057 1092
broad D,O band
-1255 1304 1342 1376 1422 1488
1580
1208-10 crn-' max 1308 1306 1349 1351 1382 1380 1425 1425 1465 1485 1484 1525 -1540 1581 1577
-
1675
1672
1306 1346 1378 1424
Assignmentb
C, T , 0-P-0 symrnetrical stretch overlap *P-0 symmetrical stretch deoxyribose-phosphate deoxyribose deoxyribose C-0 stretch C-0 stretch 0--0 symmetrical stretch deoxyribose-phosphate base C-N stretch A G, A
1578
A T, A , G A, G deoxyribose G,A A G ,C G, A
1673
C=O
1482 1525
stretch
In the presence of 0.01-0.15 M NaC1. Ref. 39.
poly(L-arginine), poly(L-valine), and poly(L-leucine) has been dis~ussed.~~,~~,~~] The assignment of the location of the band maxima summarized in Table I11 was based on the studies of Small and P e t i c ~ l a s . ~ The ~ important 830-835 cm-l band or shoulder that characterizes the vibrations of the DNA phosphate diester P-0 single bonds in the geometry of the B ~ o n f o r m a t i o n ~is~ ~ present ~0 in all the complexes we have examined (Figs. 7-9). The corresponding DNA bands or sRNA and poly(A).poly(U) that are known to be in the A conformation are observed a t 811-814 cm-1.39-41 These findings clearly rule out the A conformation of DNA in DNA-protamine complexes.
DISCUSSION The Inhibition of B
-
-+
A Transition by Protamine
One of the most important results of the present investigation is the suppression of the B A transition of DNA, which normally occurs as a function of relative humidity at controlled sodium chloride content.13J5v42 In the presence of protamine, the B A transition of DNA is suppressed at lower humidities under appropriate salt content. In-
-
700
HERSKOVITS AND BRAHMS
DNA
I
I
I
I
I
I
El
DNA - Protamine
f
Fig. 7. b a n spectra. (a) Calf thymus DNA alone in DzO, 0.15 M NaCl, pH 7.4. (b) DNA-protaminecomplex (protein-to-DNAratio = 0.7 w/w)in DzO, 0.15 M NaCI, pH 7.3. (c) Protamine chloride in DzO, 0.15 M NaC1, pH 7.6. Temperature 25O-28OC.
701
DNA-PROTAMINE COMPLEXES
uiy/,L;v I
g
loo0
no0
1200
I
if
1ooo
mo
m-'
Fig. 8. Raman spectra of DNA-clupeine complex (protein-to-DNA ratio = 0.7 w/w). (a) In water, 0.15 M NaCl, pH 7. (b) In DzO, 0.15 M NaCl, pH 7. (c) Clupeine sulfate (c = 3%)in DzO, 0.15 M NaCI, pH 7.6. Temperature 25O-28OC.
stead, one finds that the modified B conformation is stabilized and is found to persist down to about 45%rh (Figs. 4 and 5). These results are in qualitative agreement with X-ray diffraction studies of Subirana and Puigjaner.ll In contrast to the effects of protamine, it was found in our laboratory that poly(L-arginine) and poly(L-lysine) have no suppression at low humidity of the B A transition in DNA complexes.28 The inhibitory effect cannot be simply connected to the binding of basic arginine residues of protamine to the phosphate groups of DNA, but must reside in
-
HERSKOVITS AND BRAHMS
702
DNA
DNA-Protamine
0.3:l.O Complex
0.6:l.O Complex
1:l Complex
0
I
I
I
1N)o
1000
900
I
800 8 I cm-'
Fig. 9. The effects of increasing protamine content of DNA.protamine complexes on the conformationally sensitive 835-838-cm-' band; solvent, DzO, 0.15 M NaC1, pH 7.3-7.4, temperature 25O-28OC.
the presence of nonbasic residues. Further indication of the role of apvalue of protamine. olar residues is provided by a relatively lower T,,, DNA complexes when compared with that of poly(~-arginine).DNA~~ (Table 11). Also the changes in the CD spectra of protamine complexes (Table I1 and Fig. 6) are very small when compared with those of poly(L-lysine) complexes with DNA. The presence of apolar amino acids, such as proline, may be involved in conferring some characteristic folding of the polypeptide chains in the groove of the DNA helix proposed by Feughelman et al.9 This may be related either to the protection of this groove and the suppression of the effects of dehydration a t A transition in the comlow relative humidity on the modified B plexes, or to the steric interference of the protein in the groove with the structural change required for promoting the A form.
-
Effect of Alteration of DNA Conformation on Protamine Binding The second important effect observed is the finding that the native conformation of DNA is modified on complex formation with protamine. Both lines of evidence obtained by infrared linear dichroism on DNA-protamine films at high relative humidity, and by circular di-
DNA-PROTAMINE COMPLEXES
703
chroism in dilute solution, indicate a change of the native B form. Particularly significant is tke result of the determination of the bisector of the phosphate group OPO orientation, which indicates a rotation from about 680-70° in DNA to about 64O-67' in protamine-DNA complexes with respect to the helical axis. Similar alterations in the conformation of DNA to a modified B form (designated as B* in our other pape_rs have been observed with DNA.poly(L-lysine) and DNA. poly(L-arginine) complexes examined by ir linear dichroism.28 These results are casting new light on DNA conformation in complexes with protamine. All previously obtained X-ray diffraction data at high humidity indicate the similarity between the diffraction patterns obtained from DNA a t high relative humidity and from nucleoprotamine.299J1 The observed modification of the B form to a modified B form of DNA in complexes with protamine and also with basic polypeptides28 is in general agreement with X-ray diffraction data of WilkinslO and of Subirana and Puigjaner," which have indicated the similarity of DNA structure in these two complexes. As noted previously, the precise spatial orientation of the phosphate group is difficult to detect by X-ray diffraction methods on DNA fibers13J5s20and particularly in protein-DNA complexes. The infrared dichroism data on nucleoprotamine provide also the basis for interpreting the circular dichroism results in solution shown in Figure 6. The observed changes in the CD spectra reflect these alterations in the backbone conformation of the DNA helix and provide evidence for the persistance of modified B conformation in solution. 27928,46)
Protamine Conformation and its Location in Complexes with DNA Essentially three models have been suggested for the organization of protamine in its complex with DNA. 1) According to Wilkins and co-workersgJOprotamine is wrapped around the DNA helix in an extended (distorted) polypeptide conformation and a partly folded chain conformation; the folds occur where nonbasic residues are in pairs, so that all basic residues are able to combine with the phosphate groups of DNA. Protamine winds helically around the DNA molecule in one of its (small) grooves. 2) The model of Pardon and Richards43 is a modification of the Feughelman modelg in which protamine lies in the small groove so that charge neutralization can occur for 20 out of every 22 successive phosphate groups. Missed charges are the result of the occurrence of proline residues in the amino-acid sequence of protamine. 3) The protamine structure corresponds to a sequence of righthanded and left-handed a-helix-type conformation and its winding around the narrow groove of DNA induces its helical symmetry on the conformation of the polypeptide chain excluding those amino-acid residues that retain their preferential &-helical c o n f ~ r m a t i o n . ~In ~ this
704
HERSKOVITS AND BRAHMS
model the deviation from regularity in winding of protamine chains around the narrow groove of the DNA (due to the presence of proline and other nonbasic residues) appears to be less important. The interesting feature of this model is the requirement that not only long-range electrostatic interactions, but also hydrogen bonding and hydrophobic contacts need to be considered as factors that contribute to the stability of the protein conformation, and the cooperativity of binding to DNA. Any detailed structural model of protaminemDNA complexes will have to take into consideration the following observations. 1) The dichroism R ( I / I I ) of the complexes in the amide region is very low or absent, suggesting that a plausible site of attachment of protamine or clupeine is one of the grooves of DNA, oriented at about 50' to the polynucleotide fiber axis. 2) The observed frequencies of the amide I1 band at about 1550 cm-l (Figures 2 and 3b of DNA-clupeine complex at rh 15%) do not allow an unambiguous assignment of the protein conformation. Theoretical band assignments have been made for the a-helical conformation, extended chain conformations (parallel and antiparallel), and unordered chain conformation^^^.^^ and there is a good agreement with the observed band frequencies of the amide I and amide 11. In conclusion, there are no experimental data to accept an a-helical structure for protamine in the complex. This conclusion is also supported by the measurements of rate of deuteration of complexes by Bradbury et a1.l2 which is very fast. Finally, our infrared and laser Raman investigations did not lead to the detection of any significant changes in the frequencies of protamine amide I and I1 bands upon binding to DNA. This suggests that there is no experimental verification of the hypothesis of structural changes, such as the generation of a-helical folding upon binding to DNA.31 Evidently further investigations are necessary by circular dichroism extended to the far ultraviolet regions and infrared spectroscopy in order to obtain a better understanding of protein conformation in DNA complexes. In conclusion, the information obtained about the changes of DNA structure upon binding of protamine is rather precise, indicating a modification-of phosphate orientation particularly of the bisector of the angle OPO with respect to the helical axis. Furthermore, the protein bound to DNA inhibits the normally occurring conformational changes of DNA, stabilizing and protecting the high-humidity modified B conformation against dehydration. The authors would like to acknowledge stimulating discussions with and advice and help from Kensal E. Van Holde and Sabine Brahms. This work was in part supported by D.G.R.S.T.,Interactions Molkulaires en Biologie, Contrat 72 70387. T.T.H. was supported by a Faculty Fellowship from Fordham University, and in part by a Faculty Research Grant from Fordham University.
DNAwPROTAMINE COMPLEXES
705
References 1. Wilkins, M. H. F., Zubay, G. &Wilson, H. R. (1959) J. Mol. €501. 1,179-185. 2. Wilkins, M. H. F. & Zubay, G . (1963) J . Mol. Biol. 7,756-757. 3. Luzzati, V. & Nicolaieff, A. (1963) J . Mol. Biol. 7,142-163. 4. Bradbury, E. M., Crane-Robinson, C., Rattle, H. W. E. & Stephens, R. M. (1967) in Conformation of Biopolymers, Ramachandran, G. W., Ed., Academic, New York, pp. 583-606, Vol. 2. 5. Fasman, G. D., Schaffhausen, B., Goldsmith, L. & Adler, A. (1970) Biochemistry 9, 2814-2822. 6. .von Hippel, P. H. & McGhee, J. D. (1972) Ann. Reu. Biochem. 41,231-300. 7. Schapiro, J. T., Leng, M. & Felsenfeld, G. (1969) Biochemistry 8,3219-3232. 8. Bram, S. & Ris, H. (1971) J . Mol. Biol. 55,325-336. 9. Feughelman, M., Langridge, R., Seeds, W. E., Stokes, A. R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F., Barcklay, K. R. & Hamilton, L. D. (1955) Nature 175,834-838. 10. Wilkins, M. H. F. (1956) Cold Spring Harbor Symp. Quant. Biol. 21.75-90. 11. Subirana, J. A. & Puigjaner, L. C. (1973) in Conformation of Biological Molecules a n d Polymers-Jerusalem Symp. Quantum Chem. Biochem., pp. 645-653. 12. Bradbury, E. M., Price, W. C. & Wilkinson, G . R. (1962) J . Mol. Biol. 4,3949. 13. Pilet, J. & Brahms, J. (1972) Nature New Biol. 236,99-100. 14. Brahms, J., Pilet, J., Tran, T. P. L. & Hill, L. R. (1973) Proc. Nat. Acad. Sci. U.S. 70,3352-3355. 15. Pilet, J. & Brahms, J. (1973) Biopolymers 12,387403. 16. Olins, D. E., Olins, A. L. & von Hippel, P. H. (1968) J. Mol. Biol. 33,265-281. 17. Adler, A. J. & Fasman, G. D. (1971) J . Phys. Chem. 75,1516-1526. 18. Callanan, M. J., Carroll, W. R. & Mitchell, E. R. (1957) J . Biol. Chem. 229,279-287. 19. Huang, R. C. C., Bonner, J. & Murray, K. (1964) J. Mol. Biol. 8 , 5 6 6 4 . 20. Fuller, W., Wilkins, M. H. F., Wilson, M. R., Hamilton, L. D. & Arnott, S. (1965) J. Mol. Biol. 12,60-80. 21. Krueger, W. C. & Pschigoda, L. M, (1971) Anal. Chem. 43,675-677. 22. Raguet, J. N & Brahms, J. (1973) Biochimie 55,111-117. 23. Bradbury, E. M., Price, W. C. & Wilkinson, G. R. (1961) J . Mol. Biol. 3,301-317. 24. Shimanouchi, T., Tsuboi, M. & Kyogoku, Y. (1964) Aduan. Chem. Phys. 7,435-498. 25. Tsuboi, M. (1970) Appl. Spectrosc. Reu. 1,45-90. 26. Champeil, P., Tran, T. P. L. & Brahms, J. (1973) Biochem. Biophys. Res. Commun. 55,881-887. 27. Brahms, S., Brahms, J. & Pilet, 3. (1974) Israel J . rizern. 12,153-163. 28. iiquier, J., Pinot-Lafaix, M., Taillandier, E. & Brahms, J. (1975) Biochemistry 14, 4191-4197. 29. Yu, S. S. & Li, H. J. (1973) Biopolymers 12,2777-2788. Q 30. Ando, T. & Suzuki, K. (1967) Biochim. Biophys. A C ~ 140,375-377. 31. De Santis, P., Forni, E. & Rizzo, R. (1974) Biopolymers 13,313-326. 32. Phillips, D. M. P. (1971) Histones and Nucleohistones, Plenum, New York, pp. 47-83. 33. Inoue, S. & Ando, T. (1970) Biochemistry 9,388-394. 34. Tsuboi, M., Matsuo, K. & Tso, P. 0. P. (1966) J . Mol. Biol. 15,256-267. 35. Haynes, M., Garrett, R. A. & Gratzer, W. B. (1970) Biochemistry 9,4410-4416. 36. Li, H. J., Chang, C., Weiskopf, M., Brand, B. & Rotter, A. (1974) Biopolymers 13, 649-667. 37. Frushour, B. G. & Koenig, J. L. (1974) Biopolymers 13,455-474. 38. Yu, T . J., Lippert, J. L. & Peticolas, W. L. (1973) Biopoiqrners 12,2161-2176. 39. Small, E. W. & Peticolas, W. L. (1971) Biopolymers 10, 1377-1416. 40. Erfurth, S.c., Kiser, E. J. & Peticolas, W. L. (1972) Proc. Nat. Acad. Sci. U S . 69, 938-941.
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HERSKOVITS AND BRAHMS
41. Chen, M. C. & Thomas, Jr., G. J. (1974) Biopolymers 13,615-626. 42. Cooper, P. J. & Hamilton, L. D. (1966) J. Mol. Biol. 16,562-563. 43. Pardon, J. & Richards, B. (1973) in Subunits in Biological Systems-Biological Macromolecules Timasheff, S . N. & Fasman, G. D., Ed., M. Dekker, New York, pp. 1-70, Vol. 6. 44. Miyazawa, T. (1960) J. Chem. Phys. 32,1647-1653. 45. Krimm, S. (1962) J . Mol. Biol. 4,528-540. 46. Pilet, J., Blicharski, J. & Brahms, J. (1975) Biochemistry 14,1869-1876.
Received August 12,1975 Accepted October 13,1975
BIOPOLYMERS
Structural Investigations on DNAeProtamine Complexes T. T. HERSKOVITS* and J. BRAHMS, Institut de Biologie MolBculaire (C N.R.S.), Uniuersite de Paris V I I , Paris 5", France
Synopsis Protamine-DNA complexes in film and in solution have been investigated by means of infrared linear dichroism, ultraviolet circular dichroism, and laser Raman spectroscopy. At high relative humidity and in solution both infrared linear dichroism and ultraviolet circular dichroism indicate the presence of a modified B form of DNA (designated as B* in our other paper^^^.^^,^^). This modified B form is characterized by a change of the orienA
tation of the OPO bisector angle by about 4O with respect to the helical axis when compared to the B form of DNA. At decreasing relative humidities the same modified B form is maintained and no structural transitions B A (or B C) normally occurring in free DNA were observed. The absence of the A form in these complexes was also confirmed by laser Raman scattering studies of protamine-DNA complexes. On the basis of these results, a model of the protarnineDNA complex is proposed, which suggests that the presence of apolar amino-acid residues, and probably the folding of the polypeptide chain, is responsible for preventing the B-to-A transition; this occurs either by protecting the highhumidity modified B form against dehydration or by steric interference of this protein probably located in one of the DNA grooves.
-
-
INTRODUCTION The nucleoprotamines represent one of the simpler forms of DNAbasic-protein complexes found in eukaryotic cells. The study and characterization of the complexes formed between DNA and the protamines found in sperm nuclei of some fish and mollusks are subject to fewer difficulties of interpretation of physical data associated with the study of histone and basic synthetic polypeptide complexes of DNA and of chromatin.1-8 On the basis of X-ray diffraction studies of nucleoprotamines of sperm heads and DNA-protamine complexes, Wilkins and co-worke r ~ , ~and , ~more , ~ ~recently Subirana and Puigjaner,'l have suggested that the DNA in the nucleoprotamine complex assumes a B structure with the protamines filling the small groove of the DNA double helix. X-ray scattering studies on gels3 and early polarized infrared studies of Bradbury et a1.I2 on oriented films are largely consistent with these basic features of the organization of the nucleoprotamine particle. However, due to a less expressed orientation, the structural parameters * On leave from the Department of Chemistry, Fordham University, Bronx, N.Y. 10458. 687 0 1976 by John Wiley & Sons, Inc.
688
HERSKOVITS AND BRAHMS
of DNA and of proteins in complexes are relatively poorly known. A current method that can yield precise structural parameters of the orientation of different groups in different conformations of DNA is infrared linear d i c h r o i ~ m . ' ~ - ~ ~ One approach to the study of the conformation of DNA and proteins in nucleohistones and nucleoprotamines related to the structural organization of chromatin is to study the properties of reconstituted DNAprotein complexes and the complexes of DNA with synthetic polypepProtamines are a family of very similar proteins and they are the main constituents of DNA complexes in spermatozoa of fish. They have been named on the basis of their origin (e.g., clupeine from herring, protamine is also used to designate this protein from salmon sperm). This paper reports the results of the initial phase of our studies of the simpler DNA-protamine complexes carried out simultaneously on both oriented films and on solutions prepared from the same complexes. In addition to the results obtained by infrared dichroism methods we also report the results of circular dichroism (CD), Raman scattering, and thermal denaturation measurements.
MATERIALS A N D METHODS Materials Protamine chloride (Grade V), clupeine sulfate, and calf thymus DNA were purchased from Sigma Chemical Company. Salmon sperm DNA was obtained from Worthington; protamine sulfate, from Fluka. Spectra-pore 3 dialysis tubing, with a molecular-weight cutoff limit of 3500, purchased from Spectrum Medical Instruments (Los Angeles, Calif.), was used for most of our studies. Some of the earlier experiments including the work with 2:l complexes of protamine and DNA preparations were carried out using dialysis tubing that was pretreated using the method of Callanan et a1.18 by overnight drying a t 80OC. DNAaprotamine and DNA-clupeine complexes were prepared according to the salt gradient method of Huang et al.19 Usually 40 mg of DNA dissolved in 1.0 ml of 4.0 M NaCl, pH 7.5 was mixed with 1.0 ml of protamine or clupeine dissolved in 2.0 M NaC1, pH 7. After thorough mixing and standing for several hours or overnight, the initially turbid solutions would clear, a t which stage dialysis would be initiated. The dialysis steps used were 4 hr each against 1.5 M and 0.4 M NaCl, followed by 1hr or more against 0.3 M and 0.15 M NaCl, in the cold. In order to minimize the loss of protein during the initial stages of complex formation, calculated amounts of protamine or clupeine were also added to the dialysates (15 ml each adjusted to pH 7) so as to keep the concentrations constant both inside and outside the dialysis sack. Deuterium oxide solutions were similarly prepared, using approximately 5-ml changes of dialysate of the desired final salt content and adjusted
DNA-PROTAMINE COMPLEXES
689
to approximately 7.5 pH. (We were unable to prepare complexes of salmon sperm DNA by this method, since the initial turbidity of the DNA-protamine mixture would not clear sufficiently to any acceptable level, even after several days of standing a t room temperature.) The stock solutions of several calf thymus DNA complexes, prepared by this procedure, contained approximately 1.5-2% DNA, usually required for the preparation of oriented films and laser Raman studies. For circular dichroism (CD) and thermal denaturation T , measurements, the stock solutions were diluted by a factor 200-300 with 0.05 or 0.15 M NaCl and gentle magnetic stirring in the cold, with final pH of 6.8-7.2. DNA concentration of the complexes was based on absorbance measurements on a Cary 17 recording instrument, using the extinction coefficient of 6500 M-’ cm-I at 260 nm. Methods
Infrared Dichroism. Infrared dichroism measurements were made on a Beckman IR 9 spectrophotometer equipped with a wire grid polarizer in constant humidity cells used in this laboratory.13-15 The humidity of the oriented samples, deposited on “Irtran” plates, was controlled by use of various saturated salt solutions placed in the bottom compartment of the assembled cell in tightly fitted removable Teflon walls.13-15 The dichroic ratio R ( l / l l ) , which is equal to the absorbance ratios A ( 1) to A ( 11) corrected for water and baseline contribution at any of the bands examined, was computed from the spectra of the films recorded with the electrical vector of the polarized light first set perpendicular and then parallel to the orientation axis.I3-l5 This was accomplished by subtracting from the observed absorption a t 1090, 1230, and 1710 cm-l the contribution due to water of the film measured a t 3400 A cm-l.12J3,15 The orientation angles 8 of the OPO bisector and the 0-0 lines of the DNA phosphate groups in relation to the fiber axis were calculated using the dichroic ratios R ( 1 /11) and t h e Fraser relationship: R(1III) = (sin2 8
+ g)/(2 COG6, + g )
(1)
where the parameter g is related to the fraction f of oriented DNA, in the film, with:
f
= 1/(1
+ 3/2g)
(2)
The g and f parameters can be readily evaluated for the B or C form of DNA. Since the bases are oriented nearly perpendicular to the fiber axis of DNA in these two forms, the dichroic ratio at 1710 cm-l produced by the orientation of the bases with 8 taken as 90° gives a value for g = 1/(R1710 - 1). Once g is determined, Eq. (1) or (3) may be used to calculate the two angles associated with the dichroism of the phosphate groups a t about 1090 and 1230 cm-l:
690
HERSKOVITS AND BRAHMS
For the A form with the bases tilted at approximately 20' in relation to the fiber axis of DNA,20the g and f have been evaluated using the approximate angle of 80' for the average transition moments of the base absorption band at 1710 cm-l.15 Raman Spectra. Raman spectra were obtained on a Jarrell-Ash 25400 laser Raman spectrophotometer utilizing a Coherent Radiation model 52 argon ion laser set to 514.5 nm. The temperature of the samples was controlled by means of a brass block sample holder through which water circulated from a constant temperature bath. Capillary tubes held in the horizontal position in the middle of the specially constructed brass block were employed to contain the DNA-protamine and DNAaclupeine complexes. Circular Dichroism. Circular dichroism measurements were performed using a high-sensitivity apparatus constructed in this laboratory. The calibration of the dichrograph was carried out with a solution of 10-camphorsulfonic acid (2.19 mg/ml) taking (AE) = 2.3L21 Thermal Denaturation. Thermal denaturation measurements were made in a Shimadzu QV 50 spectrophotometer on degassed solutions of the various complexes and DNA samples investigated, as previously described.22
RESULTS Figures 1 and 2 show typical ir absorption spectra of oriented DNA. protamine and DNA-clupeine complexes measured a t different relative humidities (rh). In general, one finds all the main features of DNA spectra in the B form with the exception of two regions at 1665 and 1550 cm-l of the amides I and I1 absorption bands of protein. 1) The bands in the region of DNA base absorption 1800-1500 cm-l have been assigned to the C=C, C=N, C=O in-plane ring doublebond stretching vibrations and NH2 deformation vibration^.^^?^^ The band at 1710 cm-l is characteristic of the base-paired struct ~ r e .All ~ these ~ bands are perpendicular with respect to the orientation axis, as one finds in oriented DNA films (Fig. 3c and Refs. 12, 13,15). 2) The presence of bands at about 1230 and 1090 cm-l is assigned to antisymmetric and symmetric phosphate vibrations of DNA.
- -
In DNA the dichroism of these phosphate bands changes drastically when B A or B C transition occurs. The high-humidity B form or B family form is characterized by a predominant perpendicular dichroism of the band at 1090 -l and by the almost nondichroic band at 1230 cm-1.12J3J5 This is clearly observed in the spectrum of the DNAprotamine complex at high rh shown in Figure la. In addition, the B form (and also the C form) is characterized by the presence of an ir band
DNA-PROTAMINE COMPLEXES
1800
1800
1400
1200
H)o
69 1
800
cm-'
Fig. 1. Infrared dichroism spectra of oriented DNAeprotamine complex of 1:1 molar phosphate-to-arginine ratio with different relative humidities. (a) 98%. (b) 76%. (c) 15%. Spectra were obtained with electrical vector of the radiation parallel (- - -) and perpendicular (-) to the orientation or fiber axis of the complex; NaCl content of films 3-5% (w/w).
a t 836 cm-1.26,27 In DNA the transition to the A form at lower humidit y is clearly recognized by a parallel dichroism of the 1090-cm-' band12J5 and by the presence of two bands a t 812 and 860 cm-l shown in Figure 3c (arrows); these bands situated in the region of 750-860 cm-' are undoubtedly characteristic of the DNA backbone phosphodiester chain vibrations in the A or B geometry.27 As shown in Figures 1 and 2 none of the dna-protamine and DNA-clupeine complexes examined at different humidities exhibits the A form. The spectrum of a clupeine film is shown in Figure 3b. The protein contribution is easily observed in the region of amides 1 and I1 absorption bands at about 1660 and 1550 cm-'. The high intensity of these bands is due to a relatively greater amount of protein used for the film formation than for complexes with DNA. In complexes even at relatively lower protamine-to-DNA ratios the protein contribution can be clearly distinguished, particularly in the region of the amide band at about 1500 cm-' a t lower rh (Fig. 3a). We estimate that outside of these regions the protamine contribution to the DNA-protamine cornplexes a t about 1090,1230, and 1710 cm-l will only be 10-15% of the absorbances shown at these frequencies.
692
HERSKOVITS AND BRAHMS
cm-’
Fig. 2. Infrared dichroism spectra of oriented DNA-clupeinecomplex of 1:l molar arginine-to-DNA-phosphate ratio with different relative humidities. (a) 98%. (b) 15%. Spectra were obtained with the electrical vector of the radiation parallel (- -) and perpendicular (-) to the orientation axis of the film; NaCl content of complex 3 4 % (w/w).
-
Thus the DNA characteristic bands such as those of phosphates a t 1090 and at 1230 cm-1 and of paired bases at 1710 cm-l and their orientation with respect to the orientation axis are essentially unperturbed by the contribution of protamines. Further evidence for the conclusion that the orientations at different groups relative to the helical axis are similar in DNA and in DNA-protamine complexes are provided by X-ray diffraction measurements of Wilkins e t aL9J0 and by Subirana and Puigjanerll on fibers of DNA-protamine complexes. Figures 4 and 5 show the plots of dichroic ratios R(III1)of these three bands at 1090, 1230, and 1710 cm-l as a function of relative humidity obtained on some 1:l complexes of DNA-protamine and DNAaclupeine films. This plot of dichroism shows no contribution from the A form in
(a) Fig. 3 (continued)
DNA-PROTAMINE COMPLEXES
693
Fig. 3. Comparison of infrared spectra. (a) DNA-clupeine complex a t 5%rh (the arrow indicates the amide I1 band of protein a t about 1550 cm-'. (b) Clupeine at 15%rh. (c) DNA at 66% rh (the arrows indicate the presence of 812- and 860-cm-l bands characteristic of the A form not observed in the complexes). The spectra of DNA and DNA-clupeine complexes were obtained with the electric vector of the radiation perpendicular (-) and parallel (- - -) to the orientation axis of the film.
the 80-50% rh region, seen with the DNA films by Pilet and Brahms13-15 and almost the absence of structural transitions as a function of relative humidity. In fact Pilet and Brahms13?l5have found that the dichroism of DNA (calf thymus) films (NaC1 content 44% w/w) at 1090-cm-l band changed from perpendicular (e.g. R = 1.3) to parallel values (below unity, R ='0.8); a t the 1230-cm-' band the change in DNA conformation was reflected in an increase in R (from almost 1 to about 1.3). No such
HERSKOVITS AND BRAHMS
694
changes were found in complexes of DNA-protamine and DNALclupeine and only the B-type form was seen at different rh's. This is also confirmed by Subirana and Puigjanerll who have found by X-ray diffraction measurements on fibers of DNA-protamine complexes from three mollusk species that the high-humidity B type of conformation of DNA was also stabilized to changes in relative humidity. This is in contrast with the absence of such an inhibitory effect on structural transitions observed in this laboratory in complexes of DNA with basic polypeptides: poly(L-arginine) or poly(L-lysine).28 Table I presents structural parameters of various 1:l and 2:l complexes (molar ratios of arginine to DNA phosphate) of DNA-protamine and DNA-clupeine films obtained from analysis of the data a t the high (90-100%) rh region. On an average, the orientation OPO bisector angles of the complexes are about 4' lower than the angle of the B form of calf thymus DNA15 (particularly well expressed in 2:l complexes). This is considered as a modified B form. The measured distortion in angular orientation of the phosphate groups of DNA in the complexes in films is consistent with the observed changes in the CD spectra of the complexes in solution in the neighborhood of the 276-nm CD band (Table 11).
r.h.m"c) 47
76
90
95
I
I
I
I
I
DNA-Pmtamine,l:l
2.0
R[l/iI]
5
l.8
-
1.6
-
loo% 1
0
1.2 1.0 1.4
0.8I
a2
I
0.4
I
0.6
4 B
DNA-PROTAMINE COMPLEXES
695
r.h.
2.2
-
5
47
I
1
76869093 I
I
I
I
95
Kn
I
I
DNA-Ckrpeine.l:l
1.8 -
2.0
R[*/l,y
:
1.4
-
1.2
-
1.0
-
n
h
r 3
R1230
0.81
I
I
I
0.4
0.2
1
0.6
I
1.0
0.8
A3400 cm-1 (b) of)the logo-, 1230-, and 1710-cm-' bands Fig. 4. Plots of the dichroism ratios R (1/11 as a function of relative humidity of the films (1:l molar phosphate-to-arginine ratio) described in Figs. 1 and 2. (a) DNA-protamine. (b) DNAdupeine. Films contained in the final state 3-5% NaCl.
Changes in salt concentration of the solutiozls from which the films were cast had only minor effects on the conformationally sensitive 1230-cm-l dichroic band (Fig. 5a and b). It is significant that the dichroic ratios at this band show no obvious change as a function of relative humidity.14 Thus the presence of protamine stabilizes the modified-B-type conformation, designated as B* in our other papers.27728946 TABLE I Structural Parameters of DNA.Protamine and DNAClupeine Complexes at High Relative Humidity (90-100%)
Complex DNA.protarnine 1:l complex DNA.protamine 2:l complex DNAdupeine 1 :1 complex Calf thymus DNA
Film Parameters
Phosphate
Orientation
0(2)0(3) Line
O?O Bisector ( 6 , d (ded
NaCl Content of Film (70)
g
f
1.5-2.5 3-5 7-8 3-5
0.9- 1.2 0.63-0.68 0.71-0.84 1.4-1.7
0.36-0.43 0.42-0.44 0.44-0.49 0.28-0.32
55.0 t 57.0 f 57.6 i 54.0 i
3-5
0.95-1.15
0.36-0.42
53.1 i 0.8
67.9 i 1.0
3-5
2.2
0.23
56
69
( 0 , d (ded 0.4 0.4 0.6
0.3
~_
64.9i 67.5 t 66.9 f 64.9 t
0.9 0.3 0.8 0.6
HERSKOVITS AND BRAHMS
696
Circular Dichroism and Thermal Denaturation Studies The CD spectra of DNA and DNA-potamine and DNA-clupeine obtained by salt gradient dialysis are shown in Figure 6. The spectra show a noticeable reduction in the positive 275-nm band from 2.6 to approximately 2.0-2.2 and a relatively minor change a t the second, negative band near 245 nm. Table I1 presents a summary of the CD data observed a t these two extrema together with the effects of increasing protamine and clupeine in the complexes on the melting temperature T,. In a recent study of the properties of protarnine-DNA complexes, Yu and Li29 have reported very similar changes of the CD spectra of complexes prepared by direct mixing (i.e., decrease of the intensity of the positive band). The results of these solution studies are consistent with the notion that the bound protamine or clupeine alters or distorts the high-humidity B conformation of DNA, as suggested by our film infrared studies described before. Binding of a basic protein like protamine to DNA must occur through phosphate groups of the DNA backbone. Subtle angular readjustment of the phosphate groups is thus expected during the course of complexation with either synthetic or natural polypeptides rich in arginine. Of
r.h. 2 . 2 k 4 m - l l 15324758 l - l *
76 81
DNA-Protamine,l:l
0
2.0 1.8
R" 14 1.2
A3400
90 95 100
cm-1
(a)
Fig. 5 (continued)
%
DNA-PROTAMINE COMPLEXES
697
r.h. 5876 90 95 ‘L 9 15
3.4
32
I
DNA-Protamine,l:l
3.0
0
1.4
I
I
I
0.6
0.4
A3400
I
0.8
I
1.0
cm-’
(b) of the logo-, 1230-, and 1710-cm-’ bands Fig. 5. Plots of the dichroism ratios R (l/il) as a function of relative humidity of 1:l DNA.protamine complexes. (a) At low salt content. (b) At high salt content. The films in the final stage contained 1.5-2.5% and 9-15% NaCl (w/w).
the 30-32 peptide groups in the protamines and clupeine, 20-21 residues are arginine, with many of these groups running in clusters of four or five in the sequence.30 Complex formation of the basic peptide groups also confirms thermal TABLE I1 Circular Dichroism Parameters of DNA (Calf Thymus), DNA.Protamine and DNAClupeine Complexes “276
DNA calf thymus M NaCl, pH 6.9 5x 0.15 M NaCl DNA.protamine 1:1 complex M NaCl, pH 7.0 5x 0.15 M NaCl, pH 6.5 DNA.protamine 1 : 2 complex 5x M NaCl, p H 7.2 D N A d u p e i n e 1:1 complex M NaCl, p H 7.2 5x DNA-clupeine 1:2 complex M NaCI, pH 7.2 5x
“246
Ttn (“C)
2.55 2.58
-3.01 -2.96
65 f 0.5
2.45 2.50
-2.82 -2.85
77,74
2.22
-2.95
73-75
2.22
-2.7
72
2.0
-2.9
76
HERSKOVITS AND BRAHMS
698
f
’\
1.0
-
-2.0-
.
.
DNA-Clupemne
5.W’MNa CI
300
280
260
240
221)
300
1
I
I
280
260
240
I 220
Fig. 6. Circular dichroism spectra of DNA-protamine and DNA-clupeine complexes. (a) DNA-protamine arginine-to-phosphate molar ratio of complex: curve a-DNA alone; curve b-0.6; curve c--1.0; curve d-2.0, in 0.05 M NaCl, pH 7-7.2. (b) DNA-clupeine arginine-to-phosphate molar ratio of complex: curve a-DNA alone; curve b--1.0; curve c-2.0; in 0.05 M NaCl, pH 7.2.
stabilization of the complex. This is shown by the T m data also listed in Table 11. Shifts in the denaturation midpoint T m of the various complexes examined in this paper are roughly proportional to the amount of protamine or clupeine present in the complex. However, unlike the complexes obtained by direct m i ~ i n g , 2 ~ the , ~ l thermal -~~ profiles of the complexes prepared by salt gradient dialysis have revealed no clearly discernible biphasic characte? (results not shown). Differences in the melting behavior of various synthetic and natural polypeptide. DNA complexes have been noted by various authors and may depend on the method of
Raman Scattering Studies Laser Raman spectra of DNA and DNA-protamine and DNA-clupeine complexes obtained a t 25O-28OC on neutral DNA-salt solutions in both water and deuterium oxide solvents are shown in Figures 7 and 8. Figures 8 and 9 also present Raman spectra of deuterium oxide and water solutions of 3% protamine chloride and clupeine sulfate. Since these two sets of solutions had approximately several times the amount of protein present in the various DNA complexes studied, one would not expect clearly discernible contributions to the spectra of the complexes of any of the relatively weak Raman lines outside of the amide band in the region of 1650 cm-l. [The assignment of various Raman active bands in helical and nonhelical polypeptides such as poly(L-lysine) and
699
DNA-PROTAMINE COMPLEXES
TABLE I11 Characteristic Raman Frequencies of Calf Thymus DNA and 1:1 Arginine to Phosphate DNA.Protamine and DNA.Clupeine Complexes in Aqueous and Deuterium Oxide Salt Solutions a t 26" f 2°C (pH-pD 8.0-7.4)a Frequencies (cm-' ) in D , 0
Frequencies ( c m - ' ) in H,O
DNA. Protamine
DNA. Clupeine
DNA Alone
DNA. Protamine
DNA. Clupeine
DNA Alone
788
792
790
791
792
792
830-35
825-30
836-38
836-38
836-38
892
-885
1015 1058 1095
-1015 1059 1094
-1145 -1180 -1186 1230 -1225 1260 1260 1306 1310 1342 1342 1378 1376 1428 1422 -1465 1491 1490 -1515 1535 1581 1582 Broad H,O band 1650-60 cm-' max
-
-
a b
-830-35
898 925 972 1025 1058 1094
- 1005 1058 1095
896 922
895 -920 -965
1057 1093
1057 1092
broad D,O band
-1255 1304 1342 1376 1422 1488
1580
1208-10 crn-' max 1308 1306 1349 1351 1382 1380 1425 1425 1465 1485 1484 1525 -1540 1581 1577
-
1675
1672
1306 1346 1378 1424
Assignmentb
C, T , 0-P-0 symrnetrical stretch overlap *P-0 symmetrical stretch deoxyribose-phosphate deoxyribose deoxyribose C-0 stretch C-0 stretch 0--0 symmetrical stretch deoxyribose-phosphate base C-N stretch A G, A
1578
A T, A , G A, G deoxyribose G,A A G ,C G, A
1673
C=O
1482 1525
stretch
In the presence of 0.01-0.15 M NaC1. Ref. 39.
poly(L-arginine), poly(L-valine), and poly(L-leucine) has been dis~ussed.~~,~~,~~] The assignment of the location of the band maxima summarized in Table I11 was based on the studies of Small and P e t i c ~ l a s . ~ The ~ important 830-835 cm-l band or shoulder that characterizes the vibrations of the DNA phosphate diester P-0 single bonds in the geometry of the B ~ o n f o r m a t i o n ~is~ ~ present ~0 in all the complexes we have examined (Figs. 7-9). The corresponding DNA bands or sRNA and poly(A).poly(U) that are known to be in the A conformation are observed a t 811-814 cm-1.39-41 These findings clearly rule out the A conformation of DNA in DNA-protamine complexes.
DISCUSSION The Inhibition of B
-
-+
A Transition by Protamine
One of the most important results of the present investigation is the suppression of the B A transition of DNA, which normally occurs as a function of relative humidity at controlled sodium chloride content.13J5v42 In the presence of protamine, the B A transition of DNA is suppressed at lower humidities under appropriate salt content. In-
-
700
HERSKOVITS AND BRAHMS
DNA
I
I
I
I
I
I
El
DNA - Protamine
f
Fig. 7. b a n spectra. (a) Calf thymus DNA alone in DzO, 0.15 M NaCl, pH 7.4. (b) DNA-protaminecomplex (protein-to-DNAratio = 0.7 w/w)in DzO, 0.15 M NaCI, pH 7.3. (c) Protamine chloride in DzO, 0.15 M NaC1, pH 7.6. Temperature 25O-28OC.
701
DNA-PROTAMINE COMPLEXES
uiy/,L;v I
g
loo0
no0
1200
I
if
1ooo
mo
m-'
Fig. 8. Raman spectra of DNA-clupeine complex (protein-to-DNA ratio = 0.7 w/w). (a) In water, 0.15 M NaCl, pH 7. (b) In DzO, 0.15 M NaCl, pH 7. (c) Clupeine sulfate (c = 3%)in DzO, 0.15 M NaCI, pH 7.6. Temperature 25O-28OC.
stead, one finds that the modified B conformation is stabilized and is found to persist down to about 45%rh (Figs. 4 and 5). These results are in qualitative agreement with X-ray diffraction studies of Subirana and Puigjaner.ll In contrast to the effects of protamine, it was found in our laboratory that poly(L-arginine) and poly(L-lysine) have no suppression at low humidity of the B A transition in DNA complexes.28 The inhibitory effect cannot be simply connected to the binding of basic arginine residues of protamine to the phosphate groups of DNA, but must reside in
-
HERSKOVITS AND BRAHMS
702
DNA
DNA-Protamine
0.3:l.O Complex
0.6:l.O Complex
1:l Complex
0
I
I
I
1N)o
1000
900
I
800 8 I cm-'
Fig. 9. The effects of increasing protamine content of DNA.protamine complexes on the conformationally sensitive 835-838-cm-' band; solvent, DzO, 0.15 M NaC1, pH 7.3-7.4, temperature 25O-28OC.
the presence of nonbasic residues. Further indication of the role of apvalue of protamine. olar residues is provided by a relatively lower T,,, DNA complexes when compared with that of poly(~-arginine).DNA~~ (Table 11). Also the changes in the CD spectra of protamine complexes (Table I1 and Fig. 6) are very small when compared with those of poly(L-lysine) complexes with DNA. The presence of apolar amino acids, such as proline, may be involved in conferring some characteristic folding of the polypeptide chains in the groove of the DNA helix proposed by Feughelman et al.9 This may be related either to the protection of this groove and the suppression of the effects of dehydration a t A transition in the comlow relative humidity on the modified B plexes, or to the steric interference of the protein in the groove with the structural change required for promoting the A form.
-
Effect of Alteration of DNA Conformation on Protamine Binding The second important effect observed is the finding that the native conformation of DNA is modified on complex formation with protamine. Both lines of evidence obtained by infrared linear dichroism on DNA-protamine films at high relative humidity, and by circular di-
DNA-PROTAMINE COMPLEXES
703
chroism in dilute solution, indicate a change of the native B form. Particularly significant is tke result of the determination of the bisector of the phosphate group OPO orientation, which indicates a rotation from about 680-70° in DNA to about 64O-67' in protamine-DNA complexes with respect to the helical axis. Similar alterations in the conformation of DNA to a modified B form (designated as B* in our other pape_rs have been observed with DNA.poly(L-lysine) and DNA. poly(L-arginine) complexes examined by ir linear dichroism.28 These results are casting new light on DNA conformation in complexes with protamine. All previously obtained X-ray diffraction data at high humidity indicate the similarity between the diffraction patterns obtained from DNA a t high relative humidity and from nucleoprotamine.299J1 The observed modification of the B form to a modified B form of DNA in complexes with protamine and also with basic polypeptides28 is in general agreement with X-ray diffraction data of WilkinslO and of Subirana and Puigjaner," which have indicated the similarity of DNA structure in these two complexes. As noted previously, the precise spatial orientation of the phosphate group is difficult to detect by X-ray diffraction methods on DNA fibers13J5s20and particularly in protein-DNA complexes. The infrared dichroism data on nucleoprotamine provide also the basis for interpreting the circular dichroism results in solution shown in Figure 6. The observed changes in the CD spectra reflect these alterations in the backbone conformation of the DNA helix and provide evidence for the persistance of modified B conformation in solution. 27928,46)
Protamine Conformation and its Location in Complexes with DNA Essentially three models have been suggested for the organization of protamine in its complex with DNA. 1) According to Wilkins and co-workersgJOprotamine is wrapped around the DNA helix in an extended (distorted) polypeptide conformation and a partly folded chain conformation; the folds occur where nonbasic residues are in pairs, so that all basic residues are able to combine with the phosphate groups of DNA. Protamine winds helically around the DNA molecule in one of its (small) grooves. 2) The model of Pardon and Richards43 is a modification of the Feughelman modelg in which protamine lies in the small groove so that charge neutralization can occur for 20 out of every 22 successive phosphate groups. Missed charges are the result of the occurrence of proline residues in the amino-acid sequence of protamine. 3) The protamine structure corresponds to a sequence of righthanded and left-handed a-helix-type conformation and its winding around the narrow groove of DNA induces its helical symmetry on the conformation of the polypeptide chain excluding those amino-acid residues that retain their preferential &-helical c o n f ~ r m a t i o n . ~In ~ this
704
HERSKOVITS AND BRAHMS
model the deviation from regularity in winding of protamine chains around the narrow groove of the DNA (due to the presence of proline and other nonbasic residues) appears to be less important. The interesting feature of this model is the requirement that not only long-range electrostatic interactions, but also hydrogen bonding and hydrophobic contacts need to be considered as factors that contribute to the stability of the protein conformation, and the cooperativity of binding to DNA. Any detailed structural model of protaminemDNA complexes will have to take into consideration the following observations. 1) The dichroism R ( I / I I ) of the complexes in the amide region is very low or absent, suggesting that a plausible site of attachment of protamine or clupeine is one of the grooves of DNA, oriented at about 50' to the polynucleotide fiber axis. 2) The observed frequencies of the amide I1 band at about 1550 cm-l (Figures 2 and 3b of DNA-clupeine complex at rh 15%) do not allow an unambiguous assignment of the protein conformation. Theoretical band assignments have been made for the a-helical conformation, extended chain conformations (parallel and antiparallel), and unordered chain conformation^^^.^^ and there is a good agreement with the observed band frequencies of the amide I and amide 11. In conclusion, there are no experimental data to accept an a-helical structure for protamine in the complex. This conclusion is also supported by the measurements of rate of deuteration of complexes by Bradbury et a1.l2 which is very fast. Finally, our infrared and laser Raman investigations did not lead to the detection of any significant changes in the frequencies of protamine amide I and I1 bands upon binding to DNA. This suggests that there is no experimental verification of the hypothesis of structural changes, such as the generation of a-helical folding upon binding to DNA.31 Evidently further investigations are necessary by circular dichroism extended to the far ultraviolet regions and infrared spectroscopy in order to obtain a better understanding of protein conformation in DNA complexes. In conclusion, the information obtained about the changes of DNA structure upon binding of protamine is rather precise, indicating a modification-of phosphate orientation particularly of the bisector of the angle OPO with respect to the helical axis. Furthermore, the protein bound to DNA inhibits the normally occurring conformational changes of DNA, stabilizing and protecting the high-humidity modified B conformation against dehydration. The authors would like to acknowledge stimulating discussions with and advice and help from Kensal E. Van Holde and Sabine Brahms. This work was in part supported by D.G.R.S.T.,Interactions Molkulaires en Biologie, Contrat 72 70387. T.T.H. was supported by a Faculty Fellowship from Fordham University, and in part by a Faculty Research Grant from Fordham University.
DNAwPROTAMINE COMPLEXES
705
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41. Chen, M. C. & Thomas, Jr., G. J. (1974) Biopolymers 13,615-626. 42. Cooper, P. J. & Hamilton, L. D. (1966) J. Mol. Biol. 16,562-563. 43. Pardon, J. & Richards, B. (1973) in Subunits in Biological Systems-Biological Macromolecules Timasheff, S . N. & Fasman, G. D., Ed., M. Dekker, New York, pp. 1-70, Vol. 6. 44. Miyazawa, T. (1960) J. Chem. Phys. 32,1647-1653. 45. Krimm, S. (1962) J . Mol. Biol. 4,528-540. 46. Pilet, J., Blicharski, J. & Brahms, J. (1975) Biochemistry 14,1869-1876.
Received August 12,1975 Accepted October 13,1975
