Thermodynamic characterization of the osmolyte- and ligand-folded states of Bacillus subtilis ribonuclease P protein.
In Bacillus subtilis, P protein is the noncatalytic component of ribonuclease P (RNase P) that is critical for achieving maximal nuclease activity under physiological conditions. P protein is predominantly unfolded (D) at neutral pH and low ionic strength; however, it folds upon the addition of sulfate anions (ligands) as well as the osmolyte trimethylamine N-oxide (TMAO) [Henkels, C. H., Kurz, J. C., Fierke, C. A., and Oas, T. G. (2001) Biochemistry 40, 2777-2789]. Since the molecular mechanisms that drive protein folding for these two solutes are different, CD thermal denaturation studies were employed to dissect the thermodynamics of protein unfolding from the two folded states. A global fit of the free-energy of TMAO-folded P protein versus [TMAO] and temperature yields T(S), DeltaH(S), and DeltaC(p) of unfolding for the poorly populated, unliganded, folded state (N) in the absence of TMAO. These thermodynamic parameters were used in the fit of the data from the coupled unfolding/ligand dissociation reaction to obtain the sulfate dissociation constant (K(d)) and the DeltaH and DeltaC(p) of dissociation. These fits yielded a DeltaC(p) of protein unfolding of 826 +/- 23 cal mol(-)(1) K(-)(1) and a DeltaC(p) of 1554 +/- 29 cal mol(-)(1) K(-)(1) for the coupled unfolding and dissociation reaction (NL(2) --> D + 2L). The apparent stoichiometry of sulfate binding is two, so the DeltaC(p) increment of ligand dissociation is 363 +/- 9 cal mol(-)(1) K(-)(1) per site. Because N and NL(2) appear to be structurally similar and therefore similarly solvated using standard biophysical analyses, we attribute a substantial portion of this DeltaC(p) increment to an increase in conformational heterogeneity coincident with the NL(2) --> N + 2L transition.
Henkels
Christopher H
CH
Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710, USA.
Oas
Terrence G
TG
D001412:Bacillus subtilis / Q000201:enzymology*; D002942:Circular Dichroism; D006358:Hot Temperature; D008024:Ligands; D008744:Methylamines / Q000494:pharmacology; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D043262:Ribonuclease P / Q000737:chemistry*; D013431:Sulfates / Q000494:pharmacology; D013816:Thermodynamics
D008024:Ligands; D008744:Methylamines; D013431:Sulfates; D043262:Ribonuclease P; C005855:trimethyloxamine