7 research outputs found
Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29
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
Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory <i>trp</i> RNA-Binding Attenuation Protein
The <i>trp</i> RNA-binding attenuation protein (TRAP) assembles into
an 11-fold symmetric ring that regulates transcription and translation
of <i>trp</i>-mRNA in bacilli via heterotropic allosteric
activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic
resonance studies have revealed that Trp-induced activation coincides
with both microsecond to millisecond rigidification and local structural
changes in TRAP, the pathway of binding of the 11 Trp ligands to the
TRAP ring remains unclear. Moreover, because each of 11 bound Trp
molecules is completely surrounded by protein, its release requires
flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence
to study the kinetics of Trp binding by <i>Bacillus stearothermophilus</i> TRAP over a range of temperatures and observed well-separated kinetic
steps. These data were analyzed using nonlinear least-squares fitting
of several two- and three-step models. We found that a model with
two binding steps best describes the data, although the structural
equivalence of the binding sites in TRAP implies a fundamental change
in the time-dependent structure of the TRAP rings upon Trp binding.
Application of the two-binding step model reveals that Trp binding
is much slower than the diffusion limit, suggesting a gating mechanism
that depends on the dynamics of apo TRAP. These data also reveal that
dissociation of Trp from the second binding mode is much slower than
after the first Trp binding mode, revealing insight into the mechanism
for positive homotropic allostery, or cooperativity. Temperature-dependent
analyses reveal that both binding modes imbue increases in bondedness
and order toward a more compressed active state. These results provide
insight into mechanisms of cooperative TRAP activation and underscore
the importance of protein dynamics for ligand binding, ligand release,
protein activation, and allostery
pH Dependence of the Stress Regulator DksA
<div><p>DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that <i>Escherichia coli</i> DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity.</p></div
Δ<i>dksA</i> mutants are sensitive to acidic conditions.
<p>(A) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown overnight in rich medium at pH 7.8. Cultures were diluted 1:50 into LB medium at pH 2.5. At selected time points aliquots were taken and the percentage of survival of bacteria was determined using viable count. (B) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown at pH 7.8, followed by 2.5 hour adaptation at pH 6.5–4.5, and then diluted into LB medium at pH 3.5. Survival was determined using viable counts; the result after 2 hour incubation at pH 3.5 is shown. (C) DksA concentration remains relatively constant at low pH. Samples taken at different time points after a change in pH were analyzed using Western blotting with anti-DksA antibodies. Extract from the Δ<i>dksA</i> strain and purified DksA were loaded as controls.</p
DksA is sensitive to changes in pH.
<p>(A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α-<sup>32</sup>P]-GTP (10 μCi of 3000 Ci mmol<sup>−1</sup>) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the <i>rrnB</i> P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC<sub>50</sub>. The IC<sub>50</sub> values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe<sup>2+</sup> mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.</p
Substitution of His39 alters DksA sensitivity to pH.
<p>(A) The effect of pH on DksA variants. DksA activity and IC<sub>50</sub> calculations were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g003" target="_blank">Fig. 3A</a> with the <i>rrnB</i> P1 promoter. Experiments were performed at least three times at each pH. (B) Thermostability of DksA<sup>H39A</sup> is low and relatively insensitive to pH. Thermostability was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g004" target="_blank">Fig. 4</a>.</p
DksA structure is sensitive to pH.
<p>Two-dimensional <sup>1</sup>H-<sup>15</sup>N HSQC spectra at pH 8 (black) and 6 (red) reveal large chemical shift changes at (A) Tyr23 and (B) many other residues.</p