Interaction of protamine fragments with DNA

Interaction of protamine fragments with DNA Alejandro Arellano* Department of Chemistry, Faculty of Sciences, University of Concepcidn, Casilla 3-C, Concepcidn, Chile

Klaus Wehling and Karl G. Wagner Gesellschaft fiir Biotechnologische Forschung, Abteilung Molekularbiologie, Braunschweig, FR G (Received 22 April 1983; revised 14 May 1984) Applying the N--* 0 acyl rearrangement the herring protamine Clupein Y II was cleaved into defined fragments, in order to investigate the properties of the different segments of the protamine molecule. The interaction of the peptide fragments with DNA was studied by thermal denaturation, light scattering and in one case by X-ray diffraction. Furthermore, the labelling with fluorescein isothiocyanate allowed us to study the binding at equilibrium conditions. The data obtained were compared with those of the whole protamine molecule. The results for the different peptide fragments reflect their primary structure, i.e. their content of neutral or hydrophobic residues which interrupt the arginine clusters. The contribution of the two central proline residues and the importance of fl-turn formation within the protamine molecule is discussed. Keywords: DNA; protamine fragments;Clupein

Introduction In several species protamines displace the histones from chromatin during spermatogenesis and compact the DNA to form nucleoprotamine within the mature sperm heads ~. In this process protamines are first phosphorylated and dephosphorylated at the later stages 2. Different models have been postulated to describe the structure of the nucleoprotamine. Protamine molecules are proposed to bind in the small DNA groove with an extended conformation and with the arginines neutralizing phosphate groups on both sides of the groove 3 5. Another model postulates a rather compact protamine conformation including ~-helical segments, which bind to the large DNA groove 6. The different interaction modes are reflected in the proposals of the structure of free protamine in solution, possessing either an extended coil conformation 5'7 or helical clusters 6'8. Both models, however, allow protamine molecules to interact with more than one DNA strand, a property which is related to DNA compaction 5'6. There is, however, a further proposal 9 based on a nucleosome-like structure of nucleoprotamine, where the DNA strand binds around protamine molecules possessing helical globular conformation. Based on secondary structure prediction, Cid and Arellano 1° suggested a rather globular protamine conformation folded by fl-turns, which is consistent with most experimental results. This conformation seems conserved in different species like mammals, birds and fishes. As a further contribution to the mode of the interaction bf protamine with DNA, the present work is outlined to describe the behaviour of fragments of the protamine molecule. Clupein from herring was fragmented and the *

To whom correspondenceshould be addressed

interaction of the different peptides with DNA was studied using different methods.

Experimental To 300 mg of Clupein Y II extracted from herring (Clupea palasii) 11 and dried over NaOH, 5.25 ml H2SO 4 Suprapur (Merck) were added on ice. After stirring for 30 min, the mixture was incubated at 20°C (70 or 120 h), then cooled down to - 20°C and the protein precipitated with cold dried ether. The protein was collected by centrifugation and, after removing the ether under vacuum, dissolved in 10 ml 6 M HCI. The sample was titrated with 0.I M BaCI 2 and the precipitate filtered on filter paper. Sometimes, more than one precipitation with BaCI2 was necessary to remove the sulphate ions. The filtrate adjusted to 5 M HCI was hydrolysed for 20 h at 20°C. After neutralization with 6 M N a O H on ice, the solution was diluted with twice-distilled water to an ionic strength equal to those of the starting buffer (0.2 M or 0.4 M NaCI) of the subsequent chromatographic step on Sephadex CM 25. The peptides were eluted from the column (2.5 x 90 cm) in 50 mM sodium acetate buffer of pH 6.0 or 6.8 using an NaCI gradient. Each fraction was desalted on Amberlite CG 50 1 and, as acetate, lyophilized and stored. The purity of the fragments was tested by gel electrophoresis in 30% polyacrylamide according to Bretzel 3a. Only fragment preparations which revealed a single homogeneous band were used in the experiments described. Amino acids analysis was performed after hydrolysis in 6 M HCL for 20 h at 110°C, using a Biotronic and a Biocal amino acid analyser 12. N-terminal amino acids were determined using the dansyl method j3, or in the case of

0141-8130/84/0502494)6503.00 © 1984 autterworth & Co. (Publishers) Ltd

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Protamine fragment interaction with DNA" A. Arellano et al.

o

,

i

I

I

I

I

t

1

5

10

15

20

25

30

A B C

D AB ABC

BC BCD CD Figure 1 Scheme of a Clupein Y II molecule showing the points of cleavage and the possible fragments to be obtained

proline using Fluram ~*. The arginine content of the fragments was determined according to the Sakaguchi method t6 after hydrolysis of the peptides. The Clupein fragments were labelled with the F I T C isomer I (Sigma) as described xT. Separation of the labelled fragments from F I T C and salt was performed on QAE-Sephadex A 25. A molar absorption coefficient for the labelled fragments of 61 0001 mol -~ cm -~ at 500nm was used. The preparation of DNA of homogeneous size was performed as described in previous work 39. Three ml of calf thymus DNA (Boehringer) were diluted into 10 ml 1 M NaC1, 10 mM sodium acetate, 1 mM EDTA of pH 6.8 and sonicated on ice for 2.5 min using a Branson J 17 Sonifier at 50--60 W. After gel filtration on Sepharose 4 B with the same buffer, the fractions from the middle zone of the broad peak were collected and dialysed against 1 mM NaCI, 1 mM EDTA, 10 mM sodium acetate of pH 6.8. Hyperchromicity determined in 0.2 M N a O H was more than 309/o.The D N A nucleotide concentration was determined using a molar absorption coefficient of 6412 1 m o l - 1 c m - ~ at 260 nm ~5 DNA-protamine complexes were prepared with different arginine-nucleotide ratios under the appropriate ionic strength. Thermal denaturation measurements were performed with a Zeiss P M Q II spectrophotometer equipped with a Lauda NB-D 8/17 temperature control system. For stray-light experiments the optical density was monitored at 320 nm. Fluorimetric titrations were carried out as already described ~7 using a Schoeffel RRS 1000 fluorometer.

Iwai et al. 21 the rearrangement is selective with H2SO 4, the yield of broken serine peptide bonds is 86-91% , but only 16% for threonine residues, probably due to steric hindrance. Figure I shows a scheme of the total number of possible fragments and their nomenclature. Figure 2 shows the elution profiles obtained with two experiments of different duration of the acyl rearrangement, 120 h (a) and 70 h (b). The identification of the fragments was carried out on the basis of their amino acid composition, N-terminal analysis and fluram reactivity. The latter method allows us to identify those fragments which have the original Nterminal proline residue of Clupein Y II 15. The results are listed in Table 1. The 70 h experiment (b) produced the large N-terminal fragments ABC in high quantity and fragments A and B could not be detected. Obviously fragmentation did not occur at the threonine residue. At 120 h duration a more complete fragmentation pattern of the possible peptides is observed. The ionic exchange chromatographic separation shown in Figure 2 was performed at two different pHs. This changes the order of appearance of fragment C which elutes at pH 6.0 after fragment ABC but at pH 6.8 prior to that. Obviously not only electrostatic forces play a role in the interactions of this fragment with the column material. At the higher pH fragment C might become more hydrophobic, as it contaifis most of the hydrophobic residues of the whole protamine molecule.

Thermal denaturation studies Figure 3 shows the melting curves of calf thymus DNA complexed with the protamine fragments and in Table 2 the melting points are listed. Naked DNA shows a sigmoidal shape with a transition point which increases at higher salt concentrations due to stabilization of the DNA native structure by cations. Most of the complexes show a biphasic shape with a lower melting point (T°) attributed

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Results Fragmentation of Clupein Y II and characterization of the peptides The N---* O acyl rearrangement 1a,2o, used by Ando and Suzuki x9 to determine the sequence of protamines, takes place in the presence of concentrated H2SO4 at both serine and threonine residues. The hydrolysis of the ester bond with 5 M HCI yields protein fragments with the hydroxy-amino acids at the N-terminal. According to

250

Int. J. Biol. Macromol., 1984, Vol 6, October

£ ,I o25- ~ 4 -41 90

HO Fractions

tSO

Figure 2 Chromatographic separation on Sephadex CM 25 of the Clupein fragments. (a) After 120 h hydrolysis and elution at pH 6.0. (b) After 70 h hydrolysis and elution at pH 6.8

Protamine fragment interaction with D N A : A. Arellano et al. Table 1

A m i n o acid c o m p o s i t i o n for the different fragments

A m i n o acids

B

A

ABC

AB

Arg Pro Ser Thr Ala Val

2.30 2 . . . 0.81 1 0.98 1 . . .

3.01 0.71

3 1

13.1 12 2.3 3 0.61 1 0.53 1 0.89 1 1.98 2

4.98 0.85 0.79 1.20 -

-

-

N-terminal

Thr

Pro

Pro (Ser)

Pro

Ser (Pro) (Arg)

Pro (Ser) (Gly)

Ser

F l u r a m test

+

-

-

-

+

-

+

.

.

5 1 1 1 -

C

D

CD

7.12 7 1.60 2 0.70 1 . . 1.85 2

10.1 8 (0.8)1.2 1 . . 1.1 1 (0.21)-

17.1 15 1.76 2 1.89 2

.

. 1.20 1.89

1 2

In each column at the left arc the experimental and at the fight the theoretical numbers of residues. For the N-terminal analysis the amino acids in parenthesis represents traces. The positive sign for the Fluram test indicates the presence of primary amino groups, i.e. absence of proline at the amino end

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1984, V o l 6, O c t o b e r

251

Protamine fragment interaction with DNA: A. Arellano et al. Table 2 Melting temperatures of DNA-Clupein fragment complexes derived from the thermal denaturation experiments in Figure 3 Fragment

Arg/Nt

7-o (of)

T~ (of)

59.0 71.0 78.0 84.0

-

C

0.00 0.28 0.57 0.85

-

D

0.28 0.57 0.85

64.5 70.5 71.0

84.5 84.5 87.0

0.23 0.45 0.68

66.0

ABC

83.5

93.5

CD

0.20 0.40 0.60 0.80

61.0 62.5 66.0 68.5

95.0 88.0 90.5 92.5

to the denaturation of naked D N A and a second transition (Tin~) at higher temperatures assigned to protein-covered DNA 22"23. Interestingly the T° values assumed for the melting of naked D N A segments increase with higher fragment-DNA ratios. Obviously long-range effects also stabilize DNA segments not covered by protamine fragments 24. The irregular melting curves obtained with D N A and fragment ABC are clearly caused by the rather high light scattering contribution of these complexes (cf. Figure 4). On the other hand, D N A complexed with fragment C reveals monophasic melting curves with an increasing melting point at higher fragment-DNA ratios. Fragment C is from the middle portion of the protamine molecule (cf. Figure 1). It has seven arginine residues, i.e. only one residue less than fragment D, which shows biphasic melting curves. The seven arginine residues of fragment C are, however, interrupted by the two proline and one valine residue; this may be the reason for the different behaviour either by reducing affinity and/or the residence time of fragment C on the DNA giving rise to a kind of sliding mechanism.

Binding experiments The use of fluorescein-labelled protamine allows one to study its interaction with DNA at equilibrium conditions 17'28. Figure 5a shows the titration curve of DNA with the labelled fragment CD, the plot according to Scatchard 29 reveals a positive cooperativity (Figure 5b). Binding parameters were derived according to the concept of overlapping binding sites 17'3°. The affinity constant K~ describes the binding of the first ligand to an isolated site, Kc is the affinity constant for contiguous binding and o~the cooperativity factor. The stoichiometry parameter m corresponds to the number of DNA phosphates or nucleotides occupied by a protamine fragment molecule. Table 3 summarizes the results obtained with the protamine fragments in comparison with unfragmented Clupein Z. The data of fragment CD relative to Clupein Z at 0.3 M NaC1 reveal a strong reduction of the affinity constants K¢ and K~ and also of the stoichiometry factor m, as one would have expected as a result of the reduced arginine content of fragment CD. The fragments C and D differing only by one Arg residue were investigated at lower ionic strength. At 0.01 M NaC! the binding constant

ABC

A A A I

2

Arg/Nt

Figure 4 Light scattering of DNA in complexes with dilferent Clupein fragments. Light scattering was monitored by measuring the absorption at 320 nm in the same buffer as used for the thermal denaturation experiments. Titrations were performed with increasing arginin~DNA nucleotide ratio (Arg/Nt)

-6

a

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Int. J. Biol. Macromol., 1984, Vol 6, October

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Light scattering experiments The binding of basic peptides like oligolysines 25 or protamine 26 to sheared DNA leads to the formation of complexes of rather large size which can be monitored by light scattering. Figure 4 shows the results with the protamine fragments at low ionic strength. The fragments CD and ABC show an increase in the light scattering at about charge neutralization, i.e. near to an Arg/nucleotide ratio of about one, whereas with fragment D a slightly higher ratio is required. However, with fragment C also at a ratio higher than 2 there is no increase of light scattering. The formation of macrocomplexes with unfragmented protamines has been explained by its property to link several D N A molecules. Because of their limited size this may not hold for the protamine fragments, obviously the macrocomplexes are formed in this case due to charge neutralization with a concomitant loss of solubility 27. Fragment C, however, due to its unique character discussed above, may produce complexes with smaller size and/or higher solubility.

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F T C - pepfide (p.I)

Figure 5 (a) Titration curve of the binding of FITC-labelled fragment CD to DNA at 0.3 M NaC1. FR: fluorescence intensity of the reference obtained by reading a cuvette without DNA; Fp: fluorescence intensity of the sample with DNA; f: residual fluorescence of the FITC-labelled fragment at complete binding (amount of DNA increased by 100-fold). (b) Treatment of binding data according to the Scatchard formalism. - - , the experimental data; . . . . . , computed data

Protamine fragment interaction with DNA: A. Arellano et al.

Table 3 Bindingparameters for the interaction of DNA with Clupein fragments at different ionic strengths Fragment

INaCII

Kc

K i

09

m

(O.M-~) (laM-~) C

0.005 0.01

190 183

0.25 1.10

770 160

7.0 10.6

D

0.01 0.10

288 4.6

0.57 0.011

500 410

9.8 12.3

CD

0.20 0.25 0.30

109 33 8.7

0.24 0.15 0.076

450 220 110

13.1 10.8 7.8

Clupein Z

0.30

990

5.50

180

21.9

The valuesfor Clupein Z weretaken for comparisonfrom Willmitzer and Wagner17 Kc (contiguous binding), which is more accurate than Ki, is slightly higher for fragment D (8 Arg) than for fragment C (7 Arg). Attempts to determine the binding parameters for fragment ABC were unsuccessful, because the fluorescence of this peptide shows only about 203/0 quenching after binding to DNA. Table 3 also shows data on the dependence of binding upon the ionic strength. The positively charged ligands bind to DNA in competition with the Na ÷ cations building up a movable cloud of counterions along the DNA 3~-35. Hence, the K~ values decrease with increasing Na ÷ ions. This is the case with the fragments CD and D, whereas fragment C reveals only a slight reduction in Kc. According to the concept of Record, Lohman and de Haseth as the dependence of Kc upon the Na + ion concentration gives an estimation of the number of the Na + ions released by binding of a ligand molecule and from this value the number of ionic bonds per ligand molecule m, i.e. the stoichiometry can be calculated. The data of Table 3 resulted in m values of 7.8, 2.5 and 0.7 for the fragments CD (14 arginines), D (8 arginines) and C (7 arginines). The deviation from the expected number of ionic bonds, which should be correlated with the number of arginines within a fragment, are explained 35 by the presence of amino acid residues not apt to form ionic bonds. These residues are most numerous with fragment C and explain the strongest deviation in this case. Table 3 further shows that the cooperativity parameter 09 decreases with increasing ionic strength. This is in contrast to former results on unfragmented Clupein Z ~7 which revealed a significant increase. The high binding cooperativity of the whole protamine molecules was correlated with its tendency to link several DNA molecules and hence to produce a higher charge density in contrast to collinear binding along the same DNA strand17. The higher ionic strength was believed to favour DNA crosslinking in accord with the binding data. The different behaviour of the fragments obviously indicates a different binding mode relative to unfragmented protamine. Preliminary X-ray diffraction experiments on fibres of a complex of DNA with fragment D indicated a conformational change of the DNA and of the complex relative to a complex with unfragmented protamine 36. The pitch of the DNA decreases from 3.40 to 3.32 nm and also the distance between neighbouring DNA molecules from 2.25 to 1.99 nm. The unit cell is transformed from an hexagonal

to an orthorhombic type. This change of the DNA conformation which is in the direction ,of a C-like structure should also alter the local charge density of the DNA; moreover this transition should depend on the ionic strength as high Na + concentration would stabilize the B conformation of DNA. The contribution of hydrophobic residues of the fragments to the interaction with DNA was estimated by performing binding experiments in the presence of urea (cf. Ref. 17). The binding affinity of fragments CD is reduced by a factor of 5 in the presence of 2 M urea. Because of its low affinity similar experiments with fragment C could not be performed. The reduction in affinity by urea of fragment CD is smaller than with Clupein Z but larger than with (Arg)l 6 (data from Ref. 17); this indicates that the urea effect mainly reflects the hydrophobic contribution of the neutral amino acid residues. The cooperativity decreases in the presence of urea in the case of fragment CD as well as of (Arg)l 6 whereas it increases with Clupein Z 17. This may also indicate a different binding mode of the fragments relative to whole protamine.

Discussion The present work elucidates the different binding behaviour of the fragments relative to the whole protamine molecule. The differences are expressed in qualitative and quantitative respect. The different binding modes and affinities of the fragments would sum up to the known binding properties of the whole protamine molecule, provided, however, that there are no gross differences in the secondary structure of the fragments relative to the respective segments in the whole protamine molecule. The largest fragment thoroughly investigated is fragment CD which comprises about two-thirds of the protamine molecule and contains the highly basic Cterminal part of the protamine. This fragment behaves very similarly to the whole protamine molecule as evidenced by melting and light scattering of the complexes with DNA. The AG values derived from Kc at 25°C allow us to estimate a free energy per arginine residue (AG/Arg); the numbers are suitable to compare the electrostatic contribution of fragments and whole protamine. From the data of Table 3 AG/Arg values of 0.63 and 0.61 kcal/mol are extracted for the fragment CD and Clupein Z, respectively; these numbers indicate a very similar behaviour. The AG/Arg values are depending on the ionic strength, hence a direct comparison with the fragments D and C of Table 3, investigated at lower ionic strength, is not possible. With the help of the data of Willmitzer and Wagner 17 on the ionic strength dependence of the binding affinity of the whole protamine molecule one can extrapolate a AG/Arg value of 0.72 kcal/mol at 0.1 u NaC1. At the same ionic strength from the Kc value of Table 3 a AG/Arg value of 1.14 kcal/mol is derived for fragment D. This demonstrates the higher basic character of fragment D relative to the whole protamine. Fragment C represents the most hydrophobic part of the protamine molecule and also contains the two central proline residues. The unusual behaviour of this fragment complexed with DNA in the melting and light scattering stresses its unique property, especially when compared

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P r o t a m i n e f r a g m e n t interaction with D N A : A. A r e l l a n o et al.

with fragment D which contains only one arginine residue more than fragment C. The binding behaviour of the fragments and of whole protamine is not only determined by affinity contributions of the amino acid residues but also by the conformation of the peptides. This may be most strongly reflected in the behaviour of the central fragment C with its two proline residues. The conformational aspect may also be stressed by the fact that protamines from different animal species exhibit great similarities in their secondary structure. It was recently elaborated that these similarities may be the basis of specific r-turn formation 1°. Thus rturns may be the important elements for the formation of a specific secondary and tertiary structure, necessary for a specific interaction with the DNA and for DNA condensation. Moreover, r-turns may also be structural elements for protamine phosphorylation, a physiological important step in spermiogenesis. Properly placed serine residues within the r-turns may be accessible and recognized by the phosphorylating enzymes 3~. Concerning the present work the formation of r-turns is certainly impaired when the protamine molecule is cleaved in defined fragments. This should be kept in mind when comparing the properties of the fragments with that of the whole protamine molecule.

5 6 7 8 9 l0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Acknowledgements

27 28

This work was supported by the Deutsche Akademische Austauschdienst. 'Proyecto de Investigacirn 20.13.11 de la Direcci6n de Investigacirn, Universidad de Concepcirn'.

34 35

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