Mobility of Histidine Side Chains Analyzed with N NMR Relaxation and Cross-Correlation Data: Insight into Zinc-Finger-DNA Interactions.

Due to chemical exchange, the mobility of histidine (His) side chains of proteins is typically difficult to analyze by NMR spectroscopy. Using an NMR approach that is uninfluenced by chemical exchange, we investigated internal motions of the His imidazole NH groups that directly interact with DNA phosphates in the Egr-1 zinc-finger-DNA complex. In this approach, the transverse and longitudinal cross-correlation rates for N chemical shift anisotropy and N-H dipole-dipole relaxation interference were analyzed together with N longitudinal relaxation rates and heteronuclear Overhauser effect data at two magnetic field strengths.

NMR-based investigations into target DNA search processes of proteins.

To perform their function, transcription factors and DNA-repair/modifying enzymes must first locate their targets in the vast presence of nonspecific, but structurally similar sites on genomic DNA. Before reaching their targets, these proteins stochastically scan DNA and dynamically move from one site to another on DNA. Solution NMR spectroscopy provides unique atomic-level insights into the dynamic DNA-scanning processes, which are difficult to gain by any other experimental means.

Potential role of DNA methylation as a facilitator of target search processes for transcription factors through interplay with methyl-CpG-binding proteins.

Eukaryotic genomes contain numerous non-functional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences similar to the target sequence could substantially impede association of the transcription factor Egr-1 with its targets.

Regulation of transcription factors via natural decoys in genomic DNA.

Eukaryotic genomic DNA contains numerous high-affinity sites for transcription factors. Only a small fraction of these sites directly regulates target genes. Other high-affinity sites can serve as naturally present decoys that sequester transcription factors.

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.

Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means.

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble.

Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins.

Positive and negative impacts of nonspecific sites during target location by a sequence-specific DNA-binding protein: origin of the optimal search at physiological ionic strength.

The inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured the target search kinetics of the Egr-1 zinc-finger protein at various ionic strengths between 40 and 400 mM KCl and found the most efficient search at 150 mM KCl. We further investigated the kinetics of intersegment transfer, dissociation, and sliding of this protein on DNA at distinct concentrations of KCl.