7 research outputs found
Discrete-State Kinetics Model for NMR-Based Analysis of Protein Translocation on DNA at Equilibrium
In
the target DNA search process, sequence-specific DNA-binding
proteins first nonspecifically bind to DNA and stochastically move
from one site to another before reaching their targets. To rigorously
assess how the translocation process influences NMR signals from proteins
interacting with nonspecific DNA, we incorporated a discrete-state
kinetic model for protein translocation on DNA into the McConnell
equation. Using this equation, we simulated line shapes of NMR signals
from proteins undergoing translocations on DNA through sliding, dissociation/reassociation,
and intersegment transfer. Through this analysis, we validated an
existing NMR approach for kinetic investigations of protein translocation
on DNA, which utilizes NMR line shapes of two nonspecific DNAâprotein
complexes and their mixture. We found that, despite its use of simplistic
two-state approximation neglecting the presence of many microscopic
states, the previously proposed NMR approach provides accurate kinetic
information on the intermolecular translocations of proteins between
two DNA molecules. Interestingly, our results suggest that the same
NMR approach can also provide qualitative information about the one-dimensional
diffusion coefficient for proteins sliding on DNA
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
homeodomainâDNA 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
Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States
Basic
side chains play crucial roles in proteinâDNA interactions.
In this study, using NMR spectroscopy, we investigated the dynamics
of Arg and Lys side chains of the fruit fly Antennapedia homeodomain
in the free state and in the complex with target DNA. We measured <sup>15</sup>N relaxation for Arg and Lys side chains at two magnetic
fields, from which generalized order parameters for the cationic groups
were determined. Mobility of the R5 side chain, which makes hydrogen
bonds with a thymine base in the DNA minor groove, was greatly dampened.
Several Lys and Arg side chains that form intermolecular ion pairs
with DNA phosphates were found to retain high mobility with the order
parameter being <0.6 in the DNA-bound state. Interestingly, some
of the interfacial cationic groups in the complex were more mobile
than in the free protein. The retained or enhanced mobility of the
Arg and Lys side chains in the complex should mitigate the overall
loss of conformational entropy in the proteinâDNA association
and allow dynamic molecular recognition
Thermodynamic Additivity for Impacts of Base-Pair Substitutions on Association of the Egrâ1 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 ProteinâDNA Complexes
Intermolecular ion pairs (salt bridges)
are crucial for proteinâDNA
association. For two proteinâDNA 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-ÎŒs 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 CIPâSIP 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 proteinâDNA 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
Direct Observation of the Ion-Pair Dynamics at a ProteinâDNA Interface by NMR Spectroscopy
Ion pairing is one
of the most fundamental chemical interactions
and is essential for molecular recognition by biological macromolecules.
From an experimental standpoint, very little is known to date about
ion-pair dynamics in biological macromolecular systems. Absorption,
infrared, and Raman spectroscopic methods were previously used to
characterize dynamic properties of ion pairs, but these methods can
be applied only to small compounds. Here, using NMR <sup>15</sup>N
relaxation and hydrogen-bond scalar <sup>15</sup>Nâ<sup>31</sup>P <i>J</i>-couplings (<sup><i>h3</i></sup><i>J</i><sub>NP</sub>), we have investigated the dynamics of the
ion pairs between lysine side-chain NH<sub>3</sub><sup>+</sup> amino
groups and DNA phosphate groups at the molecular interface of the
HoxD9 homeodomainâDNA complex. We have determined the order
parameters and the correlation times for CâN bond rotation
and reorientation of the lysine NH<sub>3</sub><sup>+</sup> groups.
Our data indicate that the NH<sub>3</sub><sup>+</sup> groups in the
intermolecular ion pairs are highly dynamic at the proteinâDNA
interface, which should lower the entropic costs for proteinâDNA
association. Judging from the CâN bond-rotation correlation
times along with experimental and quantum-chemically derived <sup><i>h3</i></sup><i>J</i><sub>NP</sub> hydrogen-bond
scalar couplings, it seems that breakage of hydrogen bonds in the
ion pairs occurs on a sub-nanosecond time scale. Interestingly, the
oxygen-to-sulfur substitution in a DNA phosphate group was found to
enhance the mobility of the NH<sub>3</sub><sup>+</sup> group in the
intermolecular ion pair. This can partially account for the affinity
enhancement of the proteinâDNA association by the oxygen-to-sulfur
substitution, which is a previously observed but poorly understood
phenomenon