16 research outputs found
Physicochemical Properties of Ion Pairs of Biological Macromolecules
Ion pairs (also known as salt bridges) of electrostatically interacting cationic and anionic moieties are important for proteins and nucleic acids to perform their function. Although numerous three-dimensional structures show ion pairs at functionally important sites of biological macromolecules and their complexes, the physicochemical properties of the ion pairs are not well understood. Crystal structures typically show a single state for each ion pair. However, recent studies have revealed the dynamic nature of the ion pairs of the biological macromolecules. Biomolecular ion pairs undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. This dynamic behavior is reasonable in light of the fundamental concepts that were established for small ions over the last century. In this review, we introduce the physicochemical concepts relevant to the ion pairs and provide an overview of the recent advancement in biophysical research on the ion pairs of biological macromolecules
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
Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions
El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado.During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.National Institutes of HealthRevisión por pare
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