Sealy Center for Structural Biology and Molecular Biophysics

When transcription factors and DNA repair/modifying enzymes perform their function, these molecules must first locate their specific target sites in the vast presence of nonspecific but structurally similar sites on DNA. How do proteins scan DNA? Using NMR spectroscopy, stopped flow fluorescence spectroscopy, and other biophysical / biochemical methods along with mutagenesis, we are investigating how proteins scan DNA and locate their target sites.

How do proteins scan DNA?
To elucidate DNA-scanning mechanisms at both the molecular and atomic levels, we use various biophysical approaches. Recently we developed unique spectroscopic methods for investigations of the DNA-scanning process. We use NMR spectroscopy to characterize the DNA-scanning process at an atomic level. We also use stopped-flow fluorescence spectroscopy to study the target search kinetics at a molecular level under various conditions. Using these methodologies, we study how proteins scan DNA.

What dictates the efficiency of target DNA search?
In the nucleus, DNA concentration is extremely high (~100 mg/ml). The sequence-specific DNA-binding proteins can be effectively trapped at nonspecific sites. Extensive contact with DNA is advantageous for high stability of the specific complex, but can slow the search because the protein molecule has to break a larger number of interactions with DNA for translocation. Using biophysical methods together with mutagenesis, we are investigating what dictates the kinetic efficiency in the target DNA search.

How proteins bypass or displace obstacles on DNA?
In the nucleus, numerous proteins are bound to chromosomal DNA. These proteins may represent obstacles in the target DNA search process. In some cases, target association requires displacement of another protein covering the target. Using biophysical and biochemical approaches, we are investigating how proteins bypass or even displace other proteins on DNA before locating the target site.

How does trapping at natural decoys affect gene regulations?
Because chromosomal DNA contains billions of base pairs, there are many nonfunctional sites that are identical or very similar to the target DNA sequences for transcription factors. These spurious sites could trap transcription factors. For Egr-1, we are investigating the kinetic impact of the spurious sites, and influence of other proteins on the kinetic trap by the spurious sites. We expect that these studies will shed light on unprecedented mechanisms for regulation of transcription factors.

Can we engineer DNA scanning?
New knowledge of DNA scanning will allow us to improve kinetic properties of artificial transcription factors and DNA-modifying enzymes. Artificial zinc-finger proteins with desired DNA-binding specificity have gained popularity for artificial gene control/manipulation. In fact, human gene therapy based on the zinc-finger nuclease technology is in clinical trials. However, there are many reports suggesting that, despite their high affinity, artificial zinc-finger proteins do not have suitable kinetic properties. We aim to resolve this problem via a deeper understanding of DNA-scanning mechanisms.

Selected publications from this research project

1. Iwahara, J., Zandarashvili, L., Kemme, C.A., Esadze, A. (2018) NMR-based investigations into target DNA search processes of proteins. Methods 148, 57-66.
2. Kemme, C.A., Marquez, R., Luu, R.H., Iwahara, J. (2017) Potential role of DNA methylation as a facilitator of target search processes for transcription factors through interplay with methyl-CpG-binding proteins. Nucleic Acids Res 45, 7751-59.
3. Zandarashvili, L., Esadze, A., Vuzman, D., Kemme, C.A., Levy, Y., Iwahara, J. (2015) Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble. Proc Natl Acad Sci U S A 112, E5142-9.
4. Esadze A, Kemme CA, Kolomeisky AB, Iwahara J (2014) Nucleic Acids Res 42, 7039-46.
5. Zandarashvili L, Vuzman D, Esadze A, Takayama Y, Sahu D, Levy Y, Iwahara J (2012) Proc Natl Acad Sci U S A 109, E1724-32.

Most biochemists and biophysicists would agree that ionic interactions are extremely important for molecular association of protein and nucleic acids. But it is not well known that the ionic interactions are highly dynamic. Through entropic contribution to the binding free energy, the dynamics of the ionic interactions play an important role in DNA recognition by proteins. Funded by the National Science Foundation (NSF) and the National Institutes of Health (NIH), our group has spent a decade to study how ions and ionic moieties behave at an atomic level in protein-DNA association processes.

Counterion dynamics

Using nuclear magnetic resonance (NMR) spectroscopy as our primary tool for research, we study how counterions behave in the protein-DNA association. The large net charge on the surface of nucleic acids electrostatically attract and condense cations, creating a zone called the ion atmosphere. Experimental approaches for quantitative investigations of cations in the ion atmosphere have been developed. The counterions were found to rapidly diffuse within the ion atmosphere. Some of the counterions are released from the ion atmosphere when nucleic acids bind to proteins, neutralizing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had only indirectly been implicated by salt-concentration dependence of the equilibrium constants for molecular association. Recently, the direct detection of the release of counterions has become possible through spectroscopic observation of ions. This allows more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein-nucleic acid association.

Ion-pair dynamics

Using NMR and other biophysical methods, we also study the dynamic properties of ion pairs of protein side chains and DNA phosphates. The ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact. This state is called a contact ion pair (CIP). In the other major state, the cation and the anion are intervened by water. This state is called a solvent-separated ion pair (SIP). Transitions between CIP and SIP states rapidly occur at the molecular interfaces. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Compared to Lys side chains, Arg side chains exhibit a higher propensity to directly interact with nucleotide bases, partly due to stronger cation-p interactions and a smaller desolvation energy. Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play thermodynamic and kinetic roles in protein-nucleic acid interactions.

Selected publications from this research project

Pletka, C.C., Nepravishta, R., Iwahara, J. (2020) Detecting counterion dynamics in DNA-protein association. Angew Chem Int Ed 59, 1465-8.

Yu, B., Pettitt, B.M., Iwahara, J. (2019) Experimental evidence of solvent-separated ion pairs as metastable states in electrostatic interactions of biological macromolecules.   J Phys Chem Lett 10, 7937-41.

Esadze, A., Chen, C., Zandarashvili, L., Roy, S., Pettitt, B.M., Iwahara, J. (2016) Changes in conformational dynamics of basic side chains upon protein-DNA association. Nucleic Acids Res 44, 6961-70.

Chen, C., Esadze, A., Zandarashvili, L., Nguyen, D., Pettitt, B.M., Iwahara, J. (2015) Dynamic equilibria of short-range electrostatic interactions at molecular interfaces of protein-DNA complexes. J Phys Chem Lett 6, 2733-7.

Anderson, K.M., Esadze, A., Manoharan, M., Brüschweiler, R., Gorenstein, D.G., Iwahara, J. (2013) Direct observation of the ion-pair dynamics at a protein-DNA interface by NMR spectroscopy. J Am Chem Soc 135, 3613-9.

 

 

Nuclear Magnetic Resonance (NMR) spectroscopy is an important tool for our research. Our group is a major user of UTMB's NMR facility, where three Bruker Avance-III NMR spectrometers operated at the ¹H frequencies of 600, 750, and 800 MHz are available. The 800- and 750-MHz spectrometers are equipped with TCI (¹H/²H/¹³C/¹⁵N) cryoprobes, and the 600-MHz spectrometer is equipped with a QCI (¹H/²H/¹³C/¹⁵N/³¹P) cryoprobe. In addition to standard heteronuclear multi-dimensional NMR pulse sequences, we use our own pulse sequences developed for our research [See Software].