Conformational study of the chromosomal protein MC1 from the archaebacterium Methanosarcina barkeri

346

Biochimica et Biophysica Acta, 1038 (1990) 346-354

Elsevier BBAPRO 33640

Conformational study of the chromosomal protein MC1 from the archaebacterium Methanosarcina barkeri Marlrne Imbert

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B e r n a r d L a i n e 1, N i c o l e H e l b e c q u e 2, J e a n - P a u l M o r n o n 3, J e a n - P i e r r e H r n i c h a r t 2 a n d Pierre S a u t i r r e 1

t Unitd de Recherche Associde au Centre National de la Recherche Scientifique No. 409, Universit~ de Lille 11, lnstitut de Recherches sur le Cancer de Lille, Lille, 2 Unit~ 16 de l'lnstitut National de la Santd et de la Recherche M~dicale, Lille and 3 Groupe Cristallographie et simulations interactives des Macromolkcules Biologiques, Laboratoire de Mindralogie-cristallographie, C N R S URA 09, Unioersit~s P6 et 7, Paris (France)

(Received 14 December 1989)

Key words: Protein conformation; Chromosomal protein; (M. barkeri)

Methanogen chromosomal protein MC1 is a polypeptide of 93 amino acid residues (M r 10757) which represents the major protein associated with the DNA of the archaebacterium Methanosarcina barked and can protect DNA against thermal denaturation. The conformation of protein MC1 has been investigated by means of predictive methods, infrared spectroscopy, circular dichroism and tryptophan fluorescence studies. Protein MC1 has a low amount of a-helix but contains antiparallei fl-sheet strands. The larger hydrophobic cluster which contains tryptophan at position 61 appears buried in the protein. Addition of salts induces the unfolding of the protein and makes the tryptophan indole ring more rigid. With respect to its primary structure and its conformation, protein MC1 appears radically different from the chromosomal DNA-binding protein II (also called HU-type protein) in eubacteria.

Introduction Protein MC1, the major chromosomal protein of Methanosarcina barkeri strain MS [1], has been localized

in the DNA-rich areas on immunolabeUed cryosections of this archaebacterium [2]. This protein has also been isolated from other mesophilic or thermophilic strains of Methanosarcinaceae [3-6]. Protein MC1 can protect DNA against thermal denaturation [3] and modulates transcription [7], properties shared with the DNA-binding protein II, also called HU-type protein [8]. These properties are likely correlated with the ability of both proteins to modify DNA conformation. However, as DNA-binding protein II, protein MC1 is unable to form stable repeating structural units reminiscent of eukaryotic nucleosomes [2,9,10] and its function which * Present address: Universit6 des Sciences et Techniques de Lille, Laboratoire de Microbiologie - B~timent SN2, 59655 Villeneuve d'Ascq Cedex, France. Abbreviations: UV, ultraviolet; buffer A, 10 mM sodium phosphate (pH 6.0); MC1, methanogen chromosomal protein; HCA, hydrophobic cluster analysis. Correspondence: B. Laine, Unit6 de Recherche Associre CNRS 409, Place de Verdun, 59045 Lille Cedex, France.

remains unknown, must not be restricted to a packaging of the chromosomal DNA. Such a role has been assigned to the DNA-binding protein II of which the biological function remains poorly understood, but this protein seems to serve as an accessory factor in stimulating other protein-DNA interactions involved in DNA replication, transcription and gene regulation [8]. Protein MC1 is a polypeptide of 93 amino acid residues (M r 10 757) which is mainly characterized by a high amount of basic residues (24) and acidic residues (14) [11]. Protein MC1 also contains 28 hydrophobic residues which are often associated in doublets or triplets, but the distribution of the charged residues all along the polypeptide chain gives a marked hydrophilic character to this protein at least at the level of its primary structure. Indeed the protein MC1 hydrophobicity diagram drawn according to Kyte and Doolittle [12] does not show large hydrophobic domains as those encountered in the DNA-binding protein II [13]. In this chromosomal protein, the large hydrophobic domains play a crucial role in giving rise to a-helices and fl-sheet strands, and generating the formation of a globular core in the protein dimer which corresponds to the functional state of the DNA-binding protein II [14]. It was therefore of interest to check whether protein

0167-4838/90/'$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

347 MC1 shares secondary structure features common with those of eubacterial DNA-binding protein II. In particular, we investigated whether the spatial arrangement of apolar amino acid residues can favor the formation of hydrophobic domains which may be suitable for the initiation of fl-sheet and a-helix structures. The secondary structure of protein MC1 has been studied by means of empirical prediction methods based on amino acid sequence and by infrared spectroscopy and circular dichroism. Moreover the presence of two tryptophan residues in the protein MC1 allowed us to study fluorescence properties of this molecule which can give some insight into the exposure of the segments surrounding these chromophores. Our results dearly show that the secondary structure characteristics of protein MC1 and the structural changes induced by increasing salt concentration are different from those observed for eubacterial DNAbinding protein II.

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Methods

M Protein MC1 isolation Protein MC1 was prepared by affinity chromatography according to the procedure of Chartier et al. [1] on a DNA-cellulose column. The fraction containing protein MC1 was concentrated by adsorption at low ionic strength on a carboxymethyl-TSK column (bed volume 2 ml). Loading on the column was performed in 10 mM sodium phosphate (pH 6.0) (buffer A) containing 100 mM NaC1, and the adsorbed proteins were eluted with buffer A containing 1 M NaC1. Solid ammonium sulphate was added to the protein solution up to 80% saturation. The precipitate which contained the contaminants was discarded by centrifugation at 5000 × g for 90 rain and the protein MC1 was obtained pure in the supernatant as ascertained by polyacrylamide gel electrophoresis in the presence of SDS (Fig. 1) and in acidic conditions (data not shown). Protein MC1 was desalted on a Sephadex G-25 column equilibrated and eluted with buffer A containing 20 mM NaC1 and concentrated on a carboxymethyl-TSK colunm as indicated above. The purified protein MC1 was then extensively dialyzed against buffer A containing 20 mM NaC1. The yield of protein MC1 was 2 mg per 100 g of wet cells. Preparation of protein MC1 fragments The fragment 1-64 was obtained by cleavage of protein MC1 at methionine residues, whereas the fragment 60-93 was derived from digestion of the protein MC1 with the endoproteinase Arg-C. The methods to generate and purify the fragments were as described previously [11]. Concentration of protein and fragments was determined by amino acid analyses.

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Fig. 1. Sodium dodecyl sulphate gel electrophoresis of fractions obtained during the purification of the protein MC1 by ammonium sulphate precipitation. Lane 1, fraction eluted from the DNA-cellulose column; lane 2, fraction precipitated with ammonium sulphate at 80% saturation; lane 3, fraction remaining soluble in ammonium sulphate at 80% saturation; and M, low molecular weight protein markers from Pharmacia. Samples (7/~g) were treated with 5% SDS, 2% 2-mercaptoethanol for 3 min at 100°C and run at 40 mA for 150 min on a 5-30% polyacrylamide gradient gel. Electrode buffer (Trisglyeine, pH 8.3) and gel buffer (Tris-HC1, pH 8.9) were made 0.1% in SDS. The gels were stained and destained as described in Ref. 34.

Circular dichroism Circular dichroism (CD) studies were performed with a Jobin-Yvon Mark III dichrograph in cells of 0.01-1 cm pathlength. The CD spectra of protein MC1 (120/~M) and of the fragment 1-64 (40/xM) were recorded at room temperature at various sodium fluoride or sodium chloride concentrations as indicated in legends to Figs. 4-6. Infrared spectroscopy Infrared (IR) spectra of protein MC1 were recorded with a FT Nicolet 7000 spectrophotometer, using BaF2 cells. After desalting on a Sephadex G-25 column, protein MC1 was dialyzed against water and freeze-dried. The protein was redissolved in D20 and freeze-dried again. The infrared spectra of the protein MC1 (1 mM) were recorded at room temperature at various sodium chloride concentrations in D20 as indicated in the legend to Fig. 7. Fluorescence measurements Fluorescence intensity was measured on a Jobin-Yvon JY3 spectrofluorometer in cells of 1 cm pathlength.

348 Samples (6/~M) were excited at 290 or 305 nm and the fluorescence emission was recorded between 325 and 425 nm. Maximum of fluorescence intensity was reached at 345 nm and this wavelength was chosen in the fluorescence studies of the tryptophan residues as a function of the ionic strength. Fluorescence polarization measurements and lifetime studies were conducted on a SLM 4800 spectrophotometer with cells of 1 cm pathlength using glycogen as scatterer for lifetime studies. Fluorescence excitation was at 290 nm and fluorescence emission was recorded using long pass filters (~%,t-off= 418 nm). For lifetime measurements, the excitation was polarized vertically and the emission was at 54.7 ° from the vertical. Phase and modulation lifetimes were determined at the modulation frequencies, 30 and 18 MHz. Secondary structure prediction from amino acid sequence The methods of Chou and Fasman [15], Gamier et al. [16], Sette et al. [17], and the Hydrophobic Cluster Analysis (HCA) method [18] were used to predict the secondary structure of protein MC1 from its amino acid sequence. In the method of Sette, the hydrophilicity scale of Eisenberg [19], which is the most widely used, has been applied in our study. The 'Hydrophobic Cluster Analysis' (HCA) is a method for comparing and aligning protein sequences. This method analyses longrange environment of hydrophobic residues, and considers the potential of these residues to gather in clusters and to form a-helix or fl-sheet. The secondary structures can be predicted from the shape of the clusters [18] and Bissery and Mornon (unpublished data). In the joint prediction of secondary structures, the following rules were applied: (i) the four methods were used individually, and a residue was assigned to a-helix or fl-strand conformation if this conformation was predicted by at least two methods. To be considered an a-helix, a minimum of four consecutive residues must be in a-helical conformation; to be considered fl-strand, a minimum of three consecutive residues must be in fl-strand conformation. (ii) A sequence was assigned in a fl-turn, only if both methods of Chou and Fasman [15] and of Gamier et al. [16] gave results in agreement. Results

Secondary structure analysis from amino acid sequence Among the secondary structure prediction methods, the Hydrophobic Cluster Analysis deserved particular attention because it allows an easy and rapid perception of the structured regions. Results obtained by this method are presented in Fig. 2A. Taking into account the position of proline and glycine residues which are often present in loops, protein MC1 can be segmented in 11 regions ( S 1 - S l l ) , and contains seven hydrophobic

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Fig. 2. (A) Plot of M. barkeri protein MC1 obtained by Hydrophobic Cluster Analysis. * and • represent proline and glycine residues, respectively. The hydrophobic clusters C1 to C7 are circled in bold lines. The bottom line indicates the secondary structure prediction based on size, shape and environment of hydrophobic clusters. (B) Plot of the region corresponding to cluster C4 in the M. soehngenii protein MCI-b.

clusters (C1-C7). Clusters C1, C2, C3, C5 and C6 display the typical shape and orientation of segments in fl-strand structure. The large cluster C4 may correspond to an a-helix favored by the adjacent alanine residues at positions 66 and 67. An identical shaped hydrophobic cluster also surrounded by two alanine residues corresponds to an a-helix in Sperm whale deoxymyoglobin as shown by three-dimensional structure determination (Morn•n, personal communication). However, this cluster shrinks in the middle at a tryptophan residue which is a relatively weak hydrophobic amino acid, and can be considered as a rupturing element (Morn•n, personal communication). Therefore duster C4 would rather be constituted of two fl-strands separated by a loop. The first strand includes valine at positions 55 and 57 and phenylalanine at position 58, the second strand includes the dipeptide Met64-Va165. We can note that in the variants a and b of Methanothrix soehngenii protein MC1, the corresponding sequence has a high probability to form two fl-strands separated by a loop generated by a glycine residue as predicted by secondary structure prediction algorithms [6] (Fig. 2B). The cluster C7 may form a small a-helix and within the region $3, the leucine at position 29 may form with the adjacent cluster of four alanine residues an a-hehx.

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Fig. 3. Prediction of secondary structure of M. barkeri protein MC1 from its amino acid sequence by different methods, (1) Chou and Fasman [15], (2) Gamier et al. [16], (3) Sette et al. [17], (4) Gaboriaud et al. [18] and (5) joint prediction.

A scheme of the predicted secondary structures obtained by the HCA method is presented in Fig. 3 together with the results given by three other methods and the joint prediction structure. The methods used gave results in good agreement. Several points call for some comments. In the joint prediction structure, the fl-strand 55-57 was not extended up to the residue 52 as predicted by the methods 1 and 3 because according to the method of Cid et al. [20] the two adjacent positively charged residues of lysine would interrupt this fl-strand. Indeed, this latter method indicates that the sequence 55-57 exhibits a hydrophilicity profile characteristic of a buried fl-strand. By giving a high probability that the sequence 58-66 contains a fl-strand, the HCA method is in complete disagreement with the results of the three other methods which predict an a-helix. However, the method of Sette gave to this sequence a probability to form a fl-strand only slightly lower than that to form an a-helix. Thus, the predictive methods do not allow us to define a secondary structure for this region in protein MC1 sequence. According to the secondary structure prediction algorithms, the protein MC1 would contain at least five fl-strands (residues 6-8, 17-19, 44-47, 55-57 and 7375) and possibly a sixth fl-strand (residues 63-65). On the other hand the sequence 26-32 has a high propensity to form an a-helix, whereas the sequence 88-93 exhibits a lower probability to form an a-helix. Therefore, depending on the secondary structure of the sequences 58-66 and 88-93, the a-helix content of protein MC1 ranges between 8 and 24%, and the fl-strand content ranges between 17 and 21%. It is noteworthy that protein MC1 contains numerous loops including several types of turns, these loops accounting for 30% of the secondary structure of protein MC1. From the joint

prediction algorithm, protein MC1 is mainly characterized by the presence of small structured segments separated by large loops as expected for a small protein where the hydrophobic dusters are of limited size. The largest structured segment of this molecule (residues 55-66) is encountered in its median region. Circular dichroism

In the far-UV region (200-250 nm), the CD spectrum of protein MC1 in 0.01 M sodium phosphate (pH 6.0) containing 0.05 M NaF (Fig. 4) showed a negative ÷5

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350 minimum around 200 nm. Moreover its shape and ellipticity values did not change with increasing protein concentration (from 10-200 #M). These data, together with the absence of UV absorption at 310 nm, indicate that no aggregation takes place in the conditions used in this work. The results of a CD study of protein MC1 in 0.01 M sodium phosphate (pH 6.0) in function of increasing N a F concentration are presented in Fig. 4. It can be seen that the addition of N a F induced (i) a decrease in magnitude of the negative band centered at 200 nm and (ii) from 0.35 M NaF, a red shift of this band up to 210 nm. However the ellipticity value at 222 nm - which was very low even at 0.05 M N a F - did not change. A similar result was obtained when using either NaC1 or CaC12 instead of NaF. In the near UV region (250-320 nm), the addition of N a F to protein MC1 in phosphate buffer (pH 6.0) induced dichroism at high salt concentrations (c > 0.35 M; Fig. 5). These bands arise mostly from the L-transitions of the indole chromophore, the contribution of phenylalanine being very small [21]. These results indicate that an increase in ionic strength induces a conformational change of the protein, but no main secondary structure can be evidenced

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from the spectra. In particular, the ellipticity value at 222 nm remained very small whatever the ionic strength and the salts used. In addition, no change in ellipticity could be observed when varying the p H (data not shown). Quantitative analyses of the C D spectra by the method of Chen et al. [22] indicate that a-helix only accounts for 6% of the secondary structure of the protein. These results indicate that only the sequence 26-32 would be in a-helix structure and consequently the presence of a fl-strand in the region 58-66 is the most likely. Because of the presence of four proline residues at positions 68, 72, 76 and 82, the C-terminal part of protein MC1 may present several loops which could obscure the a-helix signal at 222 nm. Therefore a C D study of the N-terminal fragment (1-64) of the protein was performed (Fig. 6). Two important results emerged from this study: (i) the ellipticity value at 222 nm was identical to that found for the intact protein and (ii) no variation of the spectrum could be observed when increasing the ionic strength. These results confirm the low content of a-helix structure in protein MC1. Infrared spectroscopy

The positions and intensities of the amide I (primarily C=O stretch) and amide II ( C - N stretch and N - H bond) bands allow the interpretation of the secondary structure of proteins [23-25]; since the amide I band at

351 about 1650 cm - t is obscured in aqueous solutions, the IR spectra of protein MC1 were performed in D20. However the use of heavy water poses the additional problem of the hydrogen-deuterium exchange resulting in a change in the intensity of the undeuterated amide II band at the same frequency as the H D O band, leading us to limit our study to the amide I domain. The spectra recorded for protein MC1 in D20 (c = 1 mM) in function of NaC1 concentration are presented in Fig. 7. The presence of vibrations at 1647 and 1670 cm -1 reflects a random coil. Peaks at 1617 and 1685 cm-1 are characteristic of the antiparallel fl-sheet structure [23]. The intensity of these vibrations decreased as the salt concentration was raised. This clearly shows the presence of antiparallel fl-sheets in the protein MC1 at low salt concentration and the unfolding of this secondary structure induced by increase of ionic strength. The band observed in the region 1635-1640 cm -1 can be attributed either to a a-helix conformation [26] or to a fl-parallel sheet, as calculated by Chirgadze [24]. Moreover, it has to be noted that the relative intensity of this band does not vary when increasing salt concentration. According to another work implicating theoretical calculations [25], the presence of a-helix would be reflected by a band near 1651 cm -1, band which is not observed here. Fluorescence Protein MC1 contains two tryptophan residues at positions 61 and 74, tryptophan-61 being located in a

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1600 1650 1700 Wavelength (rim) Fig. 7. Infrared spectra of M. barkeri protein MC1 (11 mg/ml) in D20 at varying NaC1 molarity (from 0.05-0.50 M). Bar in ordinate corresponds to 0.05 Absorbance Unit. Bands corresponding to antiparallel /]-sheet and random coil are indicated by • and *, respectively.

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solution (. . . . . . ). Fluorescence (F) in ordinate is expressed in arbitrary units.

region which constitutes a hydrophobic cluster and exhibits a high potential to be structured (sequence 55-66), whereas tryptophan-74 is located in the vicinity of two fl-turns generated by the proline residues at positions 72 and 76. A study of the fluorescence of protein MC1 has therefore been undertaken in order to get some insight into the exposure of the region surrounding the tryptophan residues. In addition to these tryptophan residues, protein MC1 contains four phenylalanine residues, the fluorescence of which is too low to give a detectable emission peak. In order to ascertain that the fluorescence observed in the case of protein MC1 only originated from tryptophan, we checked that no significant peak or shoulder was detected in the spectra around 282 nm when protein MC1 was excited at either 265 or 295 nm. The fluorescence measurements were performed on the intact protein MC1 and its C-terminal fragment (residues 60-93) which contains the two tryptophan residues, the tryptophan at position 61 being one residue from the N-terminus. First of all, the influence of oxygen on the fluorescence spectra was studied. Bubbling N 2 for 1 min through the solution of polypeptides induced an increase in the fluorescence intensity in both cases (Fig. 8). However the increase was less important for the intact protein. This observation reflects the fact that in the whole molecule both tryptophan residues are less accessible to oxygen, and are probably buried in the tertiary structure of the protein. By contrast, in the C-terminal fragment, the increase of fluorescence is

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Fig. 9. Salt effect on the fluorescence properties of M. barkeri protein MC1 and its carboxy-terminal fragment (residues 60-93). (A) The fluorescence intensity at 345 nm, expressed in arbitrary units, was plotted as a function of NaC1 molarity after bubbling N 2 and excitation at 290 rim. o, Intact protein MC1 and • , C-terminal fragment. (B) The salt induced variation of fluorescence intensity of M. barkeri protein MC1 after excitation at 290 nm (<>) and 305 nm (4,). Fluorescence intensity was measured at 345 nm and bubbling N 2 and its variation is expressed in percentage.

higher and is probably due to a greater accessibility of tryptophan-61. The effect of salt on the fluorescence properties of protein MC1 and its fragment is presented in Fig. 9. The fluorescence intensity of protein MC1 sharply increased upon salt addition up to 0.5 M NaC1 and slightly increased from 0.5-0.9 M NaC1. On the contrary, the fluorescence intensity of the C-terminal fragment of the protein was independent of the ionic strength. Since one tryptophan residue occupies an external position in the peptide, these results can be thought to reflect conformational changes of the protein when increasing salt concentration. This hypothesis was confirmed by studying variations in the degree of polar-

ization of protein MC1 when varying the ionic strength. In the case of MC1, a net change in polarization was observed upon addition of NaC1 to the solution. The observed values ( P = 0.132 at 0.01 M NaC1 and P = 0.216 at 0.90 M NaC1) were similar to the ones observed for free ( P = 0.099) and immobilized ( P = 0.210) Nacetyl-tryptophanamide. This result suggests that the indole ring becomes more rigid at high ionic strength. However, it has to be emphasized that no shift in emission wavelength was observed during these studies. The experimental values of the band-width of the fluorescence spectra and the position of the maximum are not in favor of an ordered molecule, and reflect the different environments of the tryptophan residues [27]. This is in accordance with lifetime measurements. In the present study performed at 30 MHz modulation frequency, the apparent phase lifetimes for protein MC1 (Tp=2.0 ns at 0.01 M NaCI, ~p=3.3 ns at 0.90 M NaC1) were lower than the modulation lifetimes (~'m = 2.4 ns at 0.01 M NaC1, ~'m= 3.7 ns at 0.9 M NaC1). These results lead us to the proposal that the two tryptophan residues behave differently, since, according to Lakowicz [28], for a mixture of components, the Zp and % values are no longer equal when the lifetimes of the individual components are different (the phase method selects for the shorter lifetimes and the modulation method for the longer-lived species). This was also confirmed by the fact that higher apparent phase and modulation lifetimes were obtained when working at a lower modulation frequency (18 MHz). In order to determine the kind of conformational change occurring for protein MC1, a qualitative use of energy transfer results was made. Since the extent of energy transfer between similar residues is minimized at long-wavelength excitation [29], fluorescence emission ( E ) is defined as E = E 0 after excitation at 305 nm, and as E = E 0 + E t , where E t is the energy transfer after excitation at 290 nm. The salt-induced increase of the fluorescence intensity of protein MC1 was much more important after excitation at 305 nm than after excitation at 290 nm (Fig. 9B). This indicates that energy transfer decreases upon NaC1 addition and that, in these conditions, the tryptophan residues move away from each other according to the relationship: RAD = R O ( 1 / E t - 1 ) 1/6, where RAD is the distance between donor and acceptor and R 0 is the critical transfer distance [30]. Again, the data are in favor of one tryptophan residue being buried in the 55-66 hydrophobic cluster of protein MC1. Discussion

The process to purify protein MC1 using a selective precipitation with ammonium sulphate allowed us to prepare concentrated protein solutions without any turbidity, in avoiding denaturing conditions. This

353 method has a great advantage compared with the process described previously which yielded a protein having a high tendency to aggregate. To determine the secondary structure of protein MC1 from its amino acid sequence, we have chosen methods based on different principles; (i) the influence of neighbouring amino acid residues on the conformation of one given residue (methods of Chou and Fasman [15] and of G a m i e r et al. [16]); (ii) the hydrophilicity and amphipathicity characteristics of a segment (method of Sette et al. [17]); and (iii) stereochemistry (method of Gaboriaud et al. [18]). Despite the diversity of these methods, most of the results are in good agreement, which strengthens the accuracy of the joint prediction structure. The conformation of protein MC1 is characterized by a low amount of a-helix which represents 6% of the secondary structure of the protein as determined from C D spectra. In addition, the presence of antiparallel r-sheet strands has been ascertained by infrared spectroscopy. According to the secondary structure prediction algorithms, the content of r-strands in protein MC1 ranges between 17 and 21%. The study of the accessibihty of tryptophan residues to oxygen in the protein and its carboxy-terminal fragment indicated that the tryptophan residue at position 61 is buried in a region which constitutes a hydrophobic cluster, and has a high probability of being structured. Infrared spectroscopy, circular dichroism and fluorescence studies showed that salts greatly affect the conformation of protein MC1. Addition of sodium chloride induced the unfolding of antiparallel r-sheet strands and simultaneously, a stiffening of the tryptophan indole ring. Moreover, the cluster 55-66 could play an important role in the structure of the protein and its salt-induced change. Such a role is suggested by the lack of salt-induced change of the circular dichroism spectrum of the peptide covering the sequence 1-64 in which the cluster 55-66 has been destroyed by the loss of two bulky aliphatic residues and one alanine residue. By its low amount of secondary structure and the presence of only short structured segments, the structure of protein MC1 differs significantly from that of Bacillus stearothermophilus DNA-binding protein II which has been determined by X-ray diffraction [14]. This difference is more important in the amino-terminal half which consists in the later protein of two large a-helices. With a third a-hehx located in the carboxyterminus of the molecule, the total amount of helicity of Bacillus stearothermophilus DNA-binding protein II reaches 38%. Contrary to protein MC1 which becomes unfolded by increasing ionic strength, the a-hehcal content of the DNA-binding protein II isolated from Escherichia coli and Rhizobium meliloti rises from 36 to 47% when the NaC1 concentration increases from 0.02-0.6 M as shown

by a circular dichroism study [31]. U p o n addition of potassium phosphate or sodium chloride, a large increase of a-hehx content was also observed by Searcy et al. [32] in the protein H T a from the archaebacterium Thermoplasma acidophilum which exhibits a significant amino acid sequence relationship with DNA-binding protein II from various eubacteria [14,33]. Unlike the eukaryotic histones, the protein MC1 and the DNA-binding protein II do not form stable repetitive globular structures with D N A and do not protect D N A against staphylococcal nuclease digestion, thus demonstrating that chromatin organization of methanogenic bacteria is closer to that of eubacteria than to that of eukaryotes [2]. On the other hand the protein MC1 and the DNA-binding protein II can protect D N A from thermal denaturation, but the thermal denaturation profiles obtained with these proteins differ significantly [3]. These data suggest that these proteins interact with D N A through different mechanisms in correlation with the differences of primary and secondary structures observed between the two proteins.

Acknowledgments The authors are indebted to A. Lemaire and C. Denis for their skillful technical assistance, and to M. Lestiennes for editorial assistance. This work was supported by grants from the Centre National de la Recherche Scientifique, from the P61e R6gional des Ana6robies de la R6gion N o r d / P a s - d e - C a l a i s , from the Universit6 de L I L L E II and from the Fondation pour la Recherche M6dicale.

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