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<p>Understanding the interconversion between the thermodynamically distinguishable
states present in a protein folding pathway provides not only the kinetics and energetics
of protein folding but also insights into the functional roles of these states in
biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus
subtilis (P protein) was used as a model system to elucidate the general folding/unfolding
of an intrinsically unstructured protein (IUP) both in the absence and presence of
ligands.</p><p>P protein was previously characterized as an intrinsically unstructured
protein, and it is predominantly unfolded in the absence of ligands. Addition of small
anions can induce the protein to fold. Therefore, the folding and binding are tightly
coupled. Trimethylamine-N oxide (TMAO), an osmolyte that stabilizes the unliganded
folded form of the protein, enabled us to study the folding process of P protein in
the absence of ligand. Transient stopped-flow kinetic time courses at various final
TMAO concentrations showed multiphase kinetics. Equilibrium "cotitration" experiments
were performed using both TMAO and urea to obtain a TMAO-urea titration surface of
P protein. Both kinetic and equilibrium studies show evidence of an intermediate state
in the P protein folding process. The intermediate state is significantly populated
and the folding rate constants involved in the reaction are slow relative to similar
size proteins. </p><p>NMR spectroscopy was used to characterize the structural properties
of the folding intermediate of P protein. The results indicate that the N-terminal
(residues 2-19) and C-terminal regions (residues 91-116, 118 is the last residue)
are mostly unfolded. 1H-15N HSQC NMR spectra were collected at various pH values.
The results suggest that His 22 may play a major role in the energetics of the equilibria
between the unfolded, intermediate, and native states of P protein.</p><p>Ligand-induced
folding kinetics were also investigated to elucidate the overall coupled folding and
binding mechanism of P protein and the holoenzyme assembly process. Stopped flow fluorescence
experiments were performed at various final ligand concentrations and the data were
analyzed using a minimal complexity model that included three conformational states
(unfolded, intermediate and folded) in each of three possible liganding states (0,
1 and 2 ligands). The kinetic and equilibrium model parameters that best fit the
data were used to calculate the flux through each of the six possible folding/binding
pathways. This novel flux-based analysis allows evaluation of the relative importance
of pathways in which folding precedes binding or vice versa. The results indicate
that the coupled folding and binding mechanism of P protein is strongly dependent
on ligand concentration. This conclusion can be generalized to other protein systems
for which ligand binding is coupled to conformational changes.</p>
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