EDTA-Derivatized Deoxythymidine as a Tool for Rapid Determination of Protein Binding Polarity to DNA by Intermolecular Paramagnetic Relaxation Enhancement
| Autor: | Junji Iwahara, Elizabeth C. Murphy, D. Eric Anderson, G. Marius Clore |
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| Rok vydání: | 2003 |
| Předmět: |
Stereochemistry
Metal ions in aqueous solution Cleavage (embryo) Biochemistry Catalysis Ion Metal Paramagnetism Colloid and Surface Chemistry Nuclear Magnetic Resonance Biomolecular Edetic Acid Chelating Agents Manganese Chemistry Oligonucleotide Intermolecular force Nuclear Proteins DNA General Chemistry Sex-Determining Region Y Protein DNA-Binding Proteins Crystallography visual_art Reagent visual_art.visual_art_medium Nucleic Acid Conformation Calcium Protein Binding Thymidine Transcription Factors |
| Zdroj: | Journal of the American Chemical Society. 125:6634-6635 |
| ISSN: | 1520-5126 0002-7863 |
| Popis: | EDTA-derivatized deoxythymidine (dT-EDTA), incorporated into DNA and complexed to Fe2+ in the presence of dithiothreitol, is a widely used reagent for sequence-specific cleavage of duplex DNA. Using HPLC/electrospray mass spectrometry, we show that cleavage is specific to Fe2+, and no cleavage occurs when DNA-EDTA is complexed to other metal ions such as Ca2+, Mn2+, and Fe3+ even after many days. Because dT-EDTA can be incorporated at any desired position of a synthetic oligonucleotide, DNA-EDTA is ideally suited for the measurement of intermolecular paramagnetic relaxation enhancement effects between a paramagnetic ion chelated to DNA-EDTA and a bound protein. Measurements on the SRY/DNA-EDTA complex using two double-stranded oligonucleotides bearing dT-EDTA at opposite ends of the sequence indicate that intermolecular 1HN-T2 enhancement by chelated Mn2+ can be used to readily ascertain the polarity of protein binding to DNA and to derive quantitative long-range distance information for structure refinement. In the case of the SRY-DNA complex, excellent agreement between observed and calculated 1HN-T2 paramagnetic relaxation enhancement data can be achieved with insignificant shifts in the atomic coordinates ( approximately 0.25 A for all heavy atoms) while simultaneously satisfying all other experimental restraints. A unique feature of DNA-EDTA is that the relaxation enhancement effect can be tuned by judicious choice of the paramagnetic metal ion, thereby permitting a wide range of long-range intermolecular electron-proton distances, ranging from approximately 9 to 35 A, to be probed. |
| Databáze: | OpenAIRE |
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