The acid‐induced folded state of Sac7d is the native state

Abstract

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.1 H, 15 N 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 1 H and 15 N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1 H 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.Keywords: anion binding; ANS binding; circular dichroism; hyperthermophile; molten globule; NMR; small-angle X-ray scattering; Sulfolobus Proteins are commonly destabilized by acid with maximal unfolding typically occurring near pH 2~Linderström- Lang, 1924;Tanford, 1970;Matthew et al., 1985;Yang & Honig, 1993;Oliveberg et al., 1994;Garcia-Moreno, 1995!. In many cases, a further decrease in pH, or the addition of salt, leads to a collapse of the unfolded chain into a compact "denatured" state referred to as the acid or A-state~Goto et al., 1990;Fink et al., 1994!. The A-state is stabilized by anions contributed from either acid or salt. It has been shown to exist for a diverse array of proteins ranging from nearly all a-helical to largely all b-sheet~Fink et al., 1994!. In every instance described to date, the properties of the A-state have been those of the so-called molten globule~Ptitsyn, 1987globule~Ptitsyn, , 1995Kuwajima, 1989;Fink et al., 1994; Fink, 1995!, i.e., a folded intermediate with native-like secondary structure as indicated by the far-UV CD spectrum, but an expanded structure with a radius of gyration approximately 10-20% greater than that of the native state, loss of close packing tertiary contacts as indicated by the absence of near-UV CD and NMR chemical shift dispersion, and exposure of hydrophobic patches as indicated by enhanced fluorescence of the dye ANS. T...

The role of the salt concentration, proton, and phosphate binding on the thermal stability of wild and cloned DNA-binding protein Sso7d from Sulfolobus solfataricus

“…One site involves D36, positioned between K19 and K39 on the first and third strands of the ␤-sheet and K7 and K13 on the two-strands of the ␤-ribbon. The other potential binding site is between the interface of the ␤-sheet and the C-terminus of the ␣-helix and involves E62, positioned between K65 and K66 at the helix [37][38][39]. The reported data for the thermal unfolding of SSo7d by Knapp et al [17] are in good agreement with our results.…”

Thermodynamics of DNA Binding and Distortion by the Hyperthermophile Chromatin Protein Sac7d

“…65 Both Sac7d and the homologous protein Sso7d from Sulfolobus solfataricus have been the subject of a number of structural and thermodynamic studies. [66][67][68][69][70][71][72] The functions of Sac7d and Sso7d in vivo are unknown, but they are present in large amounts in Sulfolobus chromatin. Both bind DNA as monomers without sequence specificity or cooperativity, 68 and stabilize duplex DNA against thermal denaturation.…”

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect

“…Today, specific anion stabilization of molten globules at low pH is considered to be a characteristic property of molten globules. The question then arises whether specific anion stabilization is a property only of molten globules or applies also to native proteins (see also Makhatadze et al 1998;Bedell et al 2000;Sakurai et al 2001).The mechanism by which specific anions stabilize molten globules is reduction of the net positive charge on the protein through anion binding. At pH 2, the basic residues (Lys, His, Arg) of molten globules are fully ionized whereas most of the acidic groups (Asp, Glu) are protonated.…”