Basic side chains play crucial roles in protein-DNA 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 15N 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 protein-DNA association and allow dynamic molecular recognition.
Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.
Nowadays, one of the leading causes of death is cancer and its common way of treatment is photo-or radiotherapy. To increase the efficiency of these modalities one usually uses sensitizers that make cancer cell more sensitive to UV or ionizing radiation. Modified nucleosides with high electron affinity (EA), prone to dissociative electron attachment are the examples of such compounds. Irradiation of DNA modified with the radiosensitizing nucleosides with increased EA leads to the production of radicals. These radicals are believed to induce further DNA damage after hydrogen abstraction from the sugar moiety. While the first step of damage to the modified DNA -a nucleobase radical productionseems to be understood sufficiently well, the following degradation steps -i.e. hydrogen abstraction and further reactions resulting in a ''fixed'' damage require further studies. However, in order to test new radiosensitizers or understand the DNA degradation pathways models mimicking the native DNA environment are necessary. Indeed, in the cell nucleus DNA -a molecular target of radio-or phototherapy -interacts with a number of proteins. Thus, access to specific DNA-protein complexes, seem to be necessary to fully comprehend the sensitization processes. Here, we will present how a complex of the BrdG/BrdA/BrdC or BrdU-labeled dsDNA with a covalently linked peptide (dsDNA-PEP) can mimic the sensitized DNA in its native environment and serve as a tool for the development of radio-or photosensitizers. Labeled dsDNA-PEP radiolytes and photolytes were fully characterized using the LC-MS method. Moreover, the electron affinities (AEA) and circular dichroism (CD) spectra of the labeled oligonucleotides were calculated and measured. In summary, the obtained theoretical and experimental data allowed us to better understand the processes involved in the photo-and radio-damage of DNA in a well-defined model system.
The enzyme Protein Tyrosine Phosphatase 1B (PTP1B) is widely studied due to its role in a number of important cellular signaling pathways, including those related to type 2 diabetes and certain forms of cancer. Although wild type PTP1B has 435 residues, to date, most studies of PTP1B have focused on the catalytic domain, residues 1-301 or 1-321. In this study, we present a method for purifying a construct containing residues 1-393, which includes much of the previously excluded disordered C-terminus. We also perform a kinetic assay to compare the kinetics of the 1-301 catalytic domain and our 1-393 residue construct. We find that the 1-301 and 1-393 constructs display very similar kinetic characteristics under the conditions studied.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.