On the DNA Binding Protein I1 from Bacillus stearothermophilus I. PURIFICATION, STUDIES IN SOLUTION, AND CRYSTALLIZATION*

30 S, and S dichroism the has approxi- mately 45% a-helix. The secondary structure of the Bacillus considerably more to the of increasing and concentration than the homologous protein (NS1 and from Escherichia Proton magnetic resonance experi-ments show that the protein has a well folded, compact tertiary structure. The DNA binding protein has been crystallized from several precipitants as monoclinic needles and triclinic plates. The monoclinic form dif- fracts to at least 3.5 A and oscillation data from the native crystals have been collected. The protein is able to bind to both single- and double-stranded oligodeox-yribonucleotides. Upon binding, several changes occur in the protein NMR spectrum which,may be used for further analysis of the mechanism of interaction.

In eukaryotic cells, it has been well established that the genomic DNA is compactly folded into chromatin by an interaction with specific proteins called the histones (1). The existence of a similar packaging mechanism in the prokaryotes has yet to be clearly demonstrated, but chromatin-like structures have been observed by electron microscopy (2,3). This has prompted the search for histone-like molecules in the prokaryotes and several potential candidates have been reported (4-6).
One protein in particular has emerged as an important. DNA binding protein. This small, heat-stable protein has been independently isolated from Escherichia coli by several groups and variously referred to as H factor, D factor, HU, HD, and NS. Protein NS was shown to consist of two distinct species, NS1 and NS2, and this has been verified for HU (reviewed in Ref. 6). According to a recent nomenclature system ( 6 ) , we shall refer to the protein as DNA binding protein 11. Both species from E. coli have been sequenced (7,8) and each contains 90 amino acids of which 62 are identical. In solution, the protein exists as dimers and tetramers as measured by gel filtration: the protein binds to both singleand double-stranded DNA as well as to RNA and, when bound to duplex DNA, DNA synthesis by DNA polymerase * These studies were supported by Grant SFB 9 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. is inhibited and in vivo transcription is stimulated (6).
These latter observations are consistent with a histone-like role, but more direct evidence is that the protein has the ability to fold up double-stranded DNA into structures resembling chromatin (3, 9).
DNA binding protein I1 has been isolated from a variety of prokaryotic organisms and the amino acid sequence data demonstrate that the DNA binding proteins I1 are homologous but are conserved to a lesser extent than the eukaryotic histones (10). Unlike the situation in E . coli, only one species of binding protein has been found in each of these organisms.
We report here the isolation and characterization of DNA binding protein I1 from the thermophilic bacterium Bacillus stearothermophilus.

EXPERIMENTAL PROCEDURES'
Purification of DNA brndlng Protein 11. 500 g Bacillus stearotherno hilus Cells buffer I 1 1 1 and broken in a French press in the presence of D~a s e I ( 5 uqlmll.

RESULTS AND DISCUSSION
Purification-We have observed that DNA binding protein I1 associates with ribosomal subunits and with 70 S tight couples in appreciable quantities, both in E. coli and in B. stearothermophilus.' The protein has previously been isolated, together with the ribosomal proteins, from 50 S subunits of B. stearothermophilus. A new purification procedure for ribosomes which we have adopted recently allows for a faster purification of the DNA binding protein in large quantities.
This procedure (1 1) involves the purification of ribosomes by gel filtration in the presence of 1 M NH4Cl, under which condition DNA binding protein I1 is removed from the ribosome and emerges after the ribosome peak. The protein, being one of the few basic components in the protein mixture, can be easily purified by a two-step ion exchange chromatography.
The analysis of DNA binding protein I1 from B. stearothermophilus by two-dimensional gel electrophoresis is shown in Fig. 1. The protein appears to consist of only one component.
In comparison with the two E. coli proteins (NS1 and NS2), the B. stearothermophilus protein resembles NS2 in electrophoretic behavior (Fig. Ib). The Association of DNA Binding Protein 11 with the Ribosome-We have routinely observed the presence of the DNA binding protein in preparations of 30 S and 50 S subunits and in 70 S tight couples. The amount of protein isolated from ribosomal particles was comparable to that of other ribosomal proteins, both for E. coli and for B. stearothermophilus.2 70 S, 50 S, and 30 S ribosomal particles depleted of the DNA binding protein I1 by washing with 1 M NH4Cl were tested for binding. The protein was added to the ribosomes (or subunits) in "Polymix" (11) and the mixture was fractionated on a Bio-Gel A-0.5m column. The excluded ribosomal particles were analyzed for protein content by two-dimensional gel electrophoresis. In all three cases, DNA binding protein I1 was found to be present.
Solution Properties-The molecular weight of the DNA binding protein was determined using sedimentation equilibrium in the analytical ultracentrifuge. The smallest component detected had a molecular weight of 38,000, which corresponds to that of a tetramer. No evidence for the existence of a dimeric species was found. At higher protein concentrations, components with higher molecular weight were also observed. The Stokes radius of the protein was determined by analytical gel filtration on Sephadex G-50 (supertine). A value of 2.50 nm was found using loading concentrations of 1 mg/ml or * K. Appelt  proteins have a similar spectrum with a lower a-helix content of 35% (Fig. 2). The stability of the secondary structure of the protein as a function of temperature and urea concentration was determined by following the change in ellipticity at 220 n m (Fig. 3). The secondary structure of the protein "melted" at approximately 68 "C, the inflection point in the urea denaturation was around 3.5 M urea. In both cases, the secondary structure could be completely restored by returning the protein to the original condition. In comparison, the E. coli proteins were less stable. "Melting" occurred around 55 OC and the protein was denatured at approximately 2.5 M urea.
Proton magnetic resonance at 270 MHz showed that the protein has a well defined tertiary structure (Fig. 4, a and d) as indicated by the presence of amide proton resonances (7-10 ppm), perturbed aromatic resonances (5-7.5 ppm), and ring current-shifted methyl peaks (0-1 ppm). This becomes more evident when the spectrum is compared with that of the denatured protein obtained in 6 M urea (Fig. 46).
Crystallization-Crystals of DNA binding protein 11 can be grown at room temperature using a variety of precipitants.
The best crystals are obtained from 35% 2-methyl-2,4-pentandiol in the pH range 7-9, a protein concentration of 8 mg/ml, and 25 mM phosphate buffer. The crystals reach maximum size within 3-4 days.
Two crystal forms predominate. The first grows as monoclinic needles up to 2 mm in length, but only 0.15 mm in the other two dimensions (Fig. 5a). Precession photographs (Fig.   6, a and b)  the native data to a resolution of 3.5 A from one crystal, using an Arndt-Wonacott oscillation camera. The second crystal form rarely appears spontaneously. Enigmatically, the crystals grow after attempted seeding experiments with microscopic fragments of the monoclinic needles described above. A monoclinic crystal is mechanically ground up in 35% a-methyl-2,4-pentandiol and a minimal volume of this solution transferred to a hanging drop, using growth conditions identical with those for the monoclinic needles. The second form grows, however, as thin triclinic plates (Fig. 5b) with dimensions up to 0.5 X 0.5 X 0.15 mm".
Precession photographs (Fig. 6, c and d ) show the space group to be P1 with cell dimensions a = 36 8,, b =  Nucleotide Binding-Mixtures of binding protein and oligonucleotides were fractionated by gel filtration on Sephadex G-50 and the association of the nucleotide with the protein was followed using the absorbance a t 260 nm. A substantial part of the oligonucleotide added is bound to the protein.
DeoxyribonucIeotides in the range of di-to octanucleotide bind quite well, both in the single-and in the double-stranded form. Ribonucleotides appear to be bound very weakly or not at all. The binding becomes weaker at increasing salt concentrations and i s rather low above 0.1 M NaCl.
The circular dichoroic spectrum of the protein does not change after binding of oligonucleotides, the spectrum of the complex equals the sum of the two component spectra. However, in the proton magnetic resonance spectra of complexes, changes can be observed, e.g. in the methylene resonances of arginine (3.2 ppm) and in the 0.7-0.9-ppm region (Fig. 7).