4 research outputs found
Temperature Dependence of Internal Motions of Protein Side-Chain NH<sub>3</sub><sup>+</sup> Groups: Insight into Energy Barriers for Transient Breakage of Hydrogen Bonds
Although
charged side chains play important roles in protein function,
their dynamic properties are not well understood. Nuclear magnetic
resonance methods for investigating the dynamics of lysine side-chain
NH<sub>3</sub><sup>+</sup> groups were established recently. Using
this methodology, we have studied the temperature dependence of the
internal motions of the lysine side-chain NH<sub>3</sub><sup>+</sup> groups that form ion pairs with DNA phosphate groups in the HoxD9
homeodomain–DNA complex. For these NH<sub>3</sub><sup>+</sup> groups, we determined order parameters and correlation times for
bond rotations and reorientations at 15, 22, 28, and 35 °C. The
order parameters were found to be virtually constant in this temperature
range. In contrast, the bond-rotation correlation times of the NH<sub>3</sub><sup>+</sup> groups were found to depend strongly on temperature.
On the basis of transition state theory, the energy barriers for NH<sub>3</sub><sup>+</sup> rotations were analyzed and compared to those
for CH<sub>3</sub> rotations. Enthalpies of activation for NH<sub>3</sub><sup>+</sup> rotations were found to be significantly higher
than those for CH<sub>3</sub> rotations, which can be attributed to
the requirement of hydrogen bond breakage. However, entropies of activation
substantially reduce the overall free energies of activation for NH<sub>3</sub><sup>+</sup> rotations to a level comparable to those for
CH<sub>3</sub> rotations. This entropic reduction in energy barriers
may accelerate molecular processes requiring hydrogen bond breakage
and play a kinetically important role in protein function
Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egr‑1 Zinc-Finger Protein with DNA
The transcription factor Egr-1 specifically
binds as a monomer
to its 9 bp target DNA sequence, GCGÂTÂGÂGÂGCG,
via three zinc fingers and plays important roles in the brain and
cardiovascular systems. Using fluorescence-based competitive binding
assays, we systematically analyzed the impacts of all possible single-nucleotide
substitutions in the target DNA sequence and determined the change
in binding free energy for each. Then, we measured the changes in
binding free energy for sequences with multiple substitutions and
compared them with the sum of the changes in binding free energy for
each constituent single substitution. For the DNA variants with two
or three nucleotide substitutions in the target sequence, we found
excellent agreement between the measured and predicted changes in
binding free energy. Interestingly, however, we found that this thermodynamic
additivity broke down with a larger number of substitutions. For DNA
sequences with four or more substitutions, the measured changes in
binding free energy were significantly larger than predicted. On the
basis of these results, we analyzed the occurrences of high-affinity
sequences in the genome and found that the genome contains millions
of such sequences that might functionally sequester Egr-1
Dynamic Equilibria of Short-Range Electrostatic Interactions at Molecular Interfaces of Protein–DNA Complexes
Intermolecular ion pairs (salt bridges)
are crucial for protein–DNA
association. For two protein–DNA complexes, we demonstrate
that the ion pairs of protein side-chain NH<sub>3</sub><sup>+</sup> and DNA phosphate groups undergo dynamic transitions between distinct
states in which the charged moieties are either in direct contact
or separated by water. While the crystal structures of the complexes
show only the solvent-separated ion pair (SIP) state for some interfacial
lysine side chains, our NMR hydrogen-bond scalar coupling data clearly
indicate the presence of the contact ion pair (CIP) state for the
same residues. The 0.6-ÎĽs molecular dynamics (MD) simulations
confirm dynamic transitions between the CIP and SIP states. This behavior
is consistent with our NMR order parameters and scalar coupling data
for the lysine side chains. Using the MD trajectories, we also analyze
the free energies of the CIP–SIP equilibria. This work illustrates
the dynamic nature of short-range electrostatic interactions in DNA
recognition by proteins
Residence Times of Molecular Complexes in Solution from NMR Data of Intermolecular Hydrogen-Bond Scalar Coupling
The
residence times of molecular complexes in solution are important
for understanding biomolecular functions and drug actions. We show
that NMR data of intermolecular hydrogen-bond scalar couplings can
yield information on the residence times of molecular complexes in
solution. The molecular exchange of binding partners via the breakage
and reformation of a complex causes self-decoupling of intermolecular
hydrogen-bond scalar couplings, and this self-decoupling effect depends
on the residence time of the complex. For protein–DNA complexes,
we investigated the salt concentration dependence of intermolecular
hydrogen-bond scalar couplings between the protein side-chain <sup>15</sup>N and DNA phosphate <sup>31</sup>P nuclei, from which the
residence times were analyzed. The results were consistent with those
obtained by <sup>15</sup>N<sub><i>z</i></sub>-exchange spectroscopy.
This self-decoupling-based kinetic analysis is unique in that it does
not require any different signatures for the states involved in the
exchange, whereas such conditions are crucial for kinetic analyses
by typical NMR and other methods