Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein. | LitMetric
Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan; Hiroshima Research Center for Healthy Aging, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan. Electronic address:
Published: September 2022
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Article Abstract
eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
The efficient removal of 99TcO4- from alkaline nuclear waste is vital for optimizing nuclear waste management and safeguarding the environment. However, current state-of-the-art sorbent materials are constrained by their inability to simultaneously achieve high alkali resistance, rapid adsorption kinetics, large adsorption capacity, and selectivity. In this study, we synthesized a urea-rich cationic porous organic polymer, IPM-403, which demonstrates exceptional chemical stability, ultrafast kinetics (~92% removal within 30 seconds), high adsorption capacity (664 mg/g), excellent selectivity, along with multiple-cycle recyclability (up to 7 cycles), making it highly promising for the removal of ReO4- (surrogate of 99TcO4-) from nuclear wastewater.
Helical aromatic oligoamide foldamers bearing anionic side chains that mimic the overall shape and charge surface distribution of DNA were synthesized. Their interactions with chromosomal protein Sac7d, a non-sequence-selective DNA-binder that kinks DNA, were investigated by Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Circular Dichroism spectroscopy (CD), melting curve analysis, Atomic Force Microscopy (AFM), and Nuclear Magnetic Resonance (NMR), as well as by single crystal X-ray crystallography. The foldamers were shown to bind to Sac7d better than a DNA duplex of comparable length.
Cre, a conservative site-specific tyrosine recombinase, is a powerful gene editing tool in the laboratory. Expanded applications in human health are hindered by lack of understanding of the mechanism by which Cre selectively binds and recombines its cognate sequences. This knowledge is essential for retargeting the enzyme to new sites and for mitigating effects of off-target recombination.
Unlabelled: The cat eye syndrome chromosome region candidate 2 (CECR2) protein is an epigenetic regulator involved in chromatin remodeling and transcriptional control. The CECR2 bromodomain (CECR2-BRD) plays a pivotal role in directing the activity of CECR2 through its capacity to recognize and bind acetylated lysine residues on histone proteins. This study elucidates the binding specificity and structural mechanisms of CECR2-BRD interactions with both histone and non-histone ligands, employing techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and a high-throughput peptide assay.
The immobilization of proteins onto clay surfaces has proven beneficial for pharmaceutical and environmental applications. This study examines the adsorption of sodium caseinate (Cas), an amphiphilic protein widely used in pharmaceutical formulations, onto sodium montmorillonite (Mt). Adsorption isotherms and kinetics were examined at two pHs, above and below Cas isoelectric point (IEP).
