Thermodynamics of Coupled
Folding in the Interaction
of Archaeal RNase P Proteins RPP21 and RPP29
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Abstract
We have used isothermal titration calorimetry (ITC) to
identify
and describe binding-coupled equilibria in the interaction between
two protein subunits of archaeal ribonuclease P (RNase P). In all
three domains of life, RNase P is a ribonucleoprotein complex that
is primarily responsible for catalyzing the Mg<sup>2+</sup>-dependent
cleavage of the 5′ leader sequence of precursor tRNAs during
tRNA maturation. In archaea, RNase P has been shown to be composed
of one catalytic RNA and up to five proteins, four of which associate
in the absence of RNA as two functional heterodimers, POP5–RPP30
and RPP21–RPP29. Nuclear magnetic resonance studies of the <i>Pyrococcus furiosus</i> RPP21 and RPP29 proteins in their free
and complexed states provided evidence of significant protein folding
upon binding. ITC experiments were performed over a range of temperatures,
ionic strengths, and pH values, in buffers with varying ionization
potentials, and with a folding-deficient RPP21 point mutant. These
experiments revealed a negative heat capacity change (Δ<i>C</i><sub><i>p</i></sub>), nearly twice that predicted
from surface accessibility calculations, a strong salt dependence
for the interaction, and proton release at neutral pH, but a small
net contribution from these to the excess Δ<i>C</i><sub><i>p</i></sub>. We considered potential contributions
from protein folding and burial of interfacial water molecules based
on structural and spectroscopic data. We conclude that binding-coupled
protein folding is likely responsible for a significant portion of
the excess Δ<i>C</i><sub><i>p</i></sub>.
These findings provide novel structural and thermodynamic insights
into coupled equilibria that allow specificity in macromolecular assemblies