The acid-induced folded state of Sac7d is the native state.

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.

The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA.

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.

The hyperthermophile chromosomal protein Sac7d sharply kinks DNA.

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.

Linkage of protonation and anion binding to the folding of Sac7d.

The temperature, pH, and salt dependence of the folding of recombinant Sac7d from the hyperthermophile Sulfolobus acidocaldarius is mapped using multi-dimensional differential scanning calorimetry (DSC) and folding progress surfaces followed by circular dichroism. Linkage relations are derived to explain the observed dependencies, and it is shown that the data can be explained by the linkage of at least two protonation reactions and two anion binding sites to a two-state unfolding process. Circular dichroism spectra indicate that a native-like fold is stabilized at acid pH by anion binding. An apparent binding isotherm surface (folding progress versus pH and salt) is used to obtain intrinsic chloride binding constants as a function of pH for both sites. A saddle is predicted in the folding progress surface (progress versus temperature and pH) at low salt with a minimum near pH 2 and 20 degrees C with approximately 25% of the protein folded. The position of the saddle is sensitive to the intrinsic delta C degrees of unfolding and provides a third measure of delta C degrees independent of that obtained by a Kirchoff plot of DSC data and chemical denaturation. The observed enthalpy of unfolding approaches zero near the saddle making the unfolding largely invisible to DSC under these conditions. The linkage analysis demonstrates that the delta C degrees for unfolding obtained from a Kirchoff plot of DSC data should be distinguished from the intrinsic delta C degrees of unfolding. It is shown that the discrepancy between the free energy of unfolding for Sac7d obtained by DSC and that obtained by chemical denaturation may be explained by the linkage of protonation and anion binding to protein folding. The linkage analysis demonstrates the limitations of using the delta Hcal/ delta Hvh ratio an indication of two-state unfolding.

Hyperthermophile protein folding thermodynamics: differential scanning calorimetry and chemical denaturation of Sac7d.

Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidocaldarius is shown to be stable towards acid, thermal and chemical denaturation. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 degrees C as indicated by near and far UV circular dichroism spectra. Thermal unfolding followed by differential scanning calorimetry (DSC) occurs as a reversible, two-state transition from pH 0 to 10, with a maximal Tm of 90.7 degrees C between pH 5 and 9. At pH 0 the protein unfolds with a Tm of 63.3 degrees C. Plots of the enthalpy of unfolding as a function of Tm are linear and yield an anomalously low delta Cp of 497 (+/-20) cal deg-1 mol-1 using the Kirchhoff relation. Guanidine hydrochloride and urea-induced chemical denaturation of Sac7d occur reversibly and can be followed by circular dichroism. Global non-linear regression of the chemical denaturation data constrained by DSC determined values for delta Hm and Tm yields a delta Cp of unfolding of 858 (+/-21) cal deg-1 mol-1. The higher delta Cp is in good agreement with that predicted from the buried polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable size, indicating that the packing and change in solvent-accessible surface area on unfolding are not unusual. Similarly, guanidine hydrochloride and urea m-values are in good agreement with those expected for a protein of 66 residues. Possible explanations for the difference in delta Cp determined by application of the Kirchhoff relation to DSC data and that determined by the global fit are discussed. Protein stability curves defined by either delta Cp values are similar to those observed for small mesophile proteins. Although the protein is thermally stable, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (+/-0.1) kcal mol-1 at the growth temperature of 80 degrees C. The large number of potential ion pairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monomethylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins. Additional stabilization in vivo is expected from compatible osmolytes (polyamines) and DNA-binding.