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Biochemistry. 2001 Mar 6;40(9):2777-89.

Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein.

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Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710, USA.


Ribonuclease P (RNase P) is the endoribonuclease responsible for the 5'-maturation of precursor tRNA transcripts. In bacteria, RNase P is composed of a catalytic RNA subunit and an associated protein subunit that enhances the substrate specificity of the holoenzyme. We have initiated a study of the biophysical properties of the protein subunit from Bacillus subtilis RNase P (P protein) toward the goal of understanding the thermodynamics of RNase P holoenzyme assembly. The P protein is predominantly unfolded in 10 mM sodium cacodylate at neutral pH based on circular dichroism and NMR studies and therefore has several characteristics typical of "intrinsically unstructured" proteins. Furthermore, the P protein folds to its native alpha/beta structure upon addition of various small molecule anions. Anion-induced folding is best attributed to the binding of these anions to the folded state of the protein, and a model is presented which describes the observed tightly coupled folding and binding phenomena. The P protein also undergoes a cooperative folding transition upon addition of the osmolyte trimethylamine N-oxide (TMAO). The equilibrium constant of folding (K(fold)) at 37 degrees C for the P protein was determined to be 0.0071 +/- 0.0005 using a two-state folding model to describe the TMAO titration data. Thus, the folding and binding equilibria observed in the anion-induced folding of the P protein can be uncoupled to determine the intrinsic binding affinities (K(a)'s) of the anionic ligands. Evidence that the osmolyte-induced and the ligand-induced folded conformations of the P protein are structurally similar is also presented.

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