The proteins Sac7d and Sso7d belong to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius and S. solfactaricus, respectively. These proteins are extremely stable to heat, acid and chemical agents. Sac7d binds to DNA without any particular sequence preference and thereby increases its melting temperature by approximately 40 degrees C. We have now solved and refined the crystal structure of Sac7d in complex with two DNA sequences to high resolution. The structures are examples of a nonspecific DNA-binding protein bound to DNA, and reveal that Sac7d binds in the minor groove, causing a sharp kinking of the DNA helix that is more marked than that induced by any sequence-specific DNA-binding proteins. The kink results from the intercalation of specific hydrophobic side chains of Sac7d into the DNA structure, but without causing any significant distortion of the protein structure relative to the uncomplexed protein in solution.

Sso7d and Sac7d are two small (approximately 7,000 Mr), but abundant, chromosomal proteins from the hyperthermophilic archaeabacteria Sulfolobus solfataricus and S. acidocaldarius respectively. These proteins have high thermal, acid and chemical stability. They bind DNA without marked sequence preference and increase the Tm of DNA by approximately 40 degrees C. Sso7d in complex with GTAATTAC and GCGT(iU)CGC + GCGAACGC was crystallized in different crystal lattices and the crystal structures were solved at high resolution. Sso7d binds in the minor groove of DNA and causes a single-step sharp kink in DNA (approximately 60 degrees) by the intercalation of the hydrophobic side chains of Val 26 and Met 29. The intercalation sites are different in the two complexes. Observations of this novel DNA binding mode in three independent crystal lattices indicate that it is not a function of crystal packing.

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The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C.

The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to doublestranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [