Thermodynamic Characterization of the Osmolyte- and Ligand-Folded States of Bacillus subtilis Ribonuclease P Protein ² Christopher H. Henkels and Terrence G. Oas* Department of Biochemistry, Box 3711, Duke UniVersity Medical Center, Durham, North Carolina 27710 ReceiVed March 11, 2005; ReVised Manuscript ReceiVed June 27, 2005 ABSTRACT: 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 , ΔH S , and ΔC 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 ΔH and ΔC p of dissociation. These fits yielded a ΔC p of protein unfolding of 826 ( 23 cal mol -1 K -1 and a ΔC p of 1554 ( 29 cal mol -1 K -1 for the coupled unfolding and dissociation reaction (NL 2 f D + 2L). The apparent stoichiometry of sulfate binding is two, so the ΔC 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 ΔC p increment to an increase in conformational heterogeneity coincident with the NL 2 f N + 2L transition. A central dogma in protein science asserts that a unique, well-defined tertiary structure is essential for protein function (1). Although this tenet may hold for most proteins, an emerging class of proteins do not abide by this structure- function principle. This group is known as the “natively unfolded” or “intrinsically unstructured/disordered” protein (IUP) 1 family and consists of either full-length proteins or protein domains that do not maintain fixed, compact folded structures when analyzed by various biophysical techniques (for reviews, see refs 1-5). The diversity of biological functions that IUPs carry out is large (5). However, they can be classified into five broad functional categories: entropic chains, effectors, scavengers, assemblers, and display sites (3). All of these functional classes, except entropic chains, require the presence of a binding partner to carry out their function. Furthermore, a concurrent disorder-to- order transition occurs for the majority of the IUPs upon binding their physiological target (6). IUPs therefore are exemplary of proteins whose thermodynamic balance be- tween folded and unfolded conformations favors the unfolded state. Thus, the thermodynamic coupling of ligand binding to protein folding can be considered an extreme case of “induced fit” (7). One potential consequence of this ther- modynamic linkage is the formation of unique interaction surfaces as the favorable binding free energy can overcome the unfavorable folding free energy to lead to complex formation (3, 6). In addition to the formation of complementary surfaces with marginal stability, other possible functional advantages for IUPs have been advanced. These include (i) the regu- lation of cellular activity via targeted proteolytic degradation (1); (ii) the control of macromolecular assembly (8); (iii) the creation of extensive interaction surfaces that cannot otherwise be obtained with a compact protein (3); (iv) the potential to bind targets with a faster association rate (9); and (v) the ability to bind multiple ligands due to the inherent structural plasticity of the intrinsically disordered protein (10). Although speculative in nature, there are some intrigu- ing thermodynamic implications of these putative IUP functions. For example, structural adaptivity of the poorly populated folded conformations of an IUP implies extensive structural heterogeneity in this thermodynamically unfavored folded ensemble. Certain ligands and/or stabilizing solutes ² Supported by National Institutes of Health grants GM45322 (T.G.O.). C.H.H. was supported in part by NIH Training Grant GM08487. * To whom correspondence should be addressed. Tel: (919) 684- 4363. Fax: (919) 681-8862. E-mail: oas@biochem.duke.edu. 1 Abbreviations: CD, circular dichroism; CHS, concentration at zero enthalpy and entropy; DSC, differential scanning calorimetry; GuanHCl, guanidinium hydrochloride; HSQC, heteronuclear single quantum coherence; IUP, intrinsically unstructured protein; L or lig, ligand; p21, p21-activated kinase; PFG-NMR, pulsed-field gradient nuclear magnetic resonance; per, perturbant; pI, isoelectric point; P protein, protein subunit of Bacillus subtilis ribonuclease P; pre-tRNA, precursor tRNA; RNase P, ribonuclease P; T o, reference temperature; THS, temperature at zero enthalpy or entropy; TMAO, trimethylamine N-oxide; WASP, Wiskott-Aldrich syndrome protein. 13014 Biochemistry 2005, 44, 13014-13026 10.1021/bi0504613 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005