Nucleotide binding kinetics and conformational change analysis of tissue transglutaminase with switchSENSE.

Analytical Biochemistry
Regina StafflerFriederike M Möller

Abstract

Function, activity, and interactions of proteins crucially depend on their three-dimensional structure and are often regulated by effector binding and environmental changes. Tissue transglutaminase (Transglutaminase 2, TG2) is a multifunctional protein, allosterically regulated by nucleotides and Ca2+ ions, which trigger opposing conformational changes. Here we introduce switchSENSE as a versatile tool for TG2 characterization and provide novel insights into protein conformation as well as analyte binding kinetics. For the first time, we succeeded in measuring the kinetic rate constants and affinities (kon, koff, KD) for guanosine nucleotides (GMP, GDP, GTP, GTPγS). Further, the conformational changes induced by GDP, Ca2+ and the covalent inhibitor Z-DON were observed by changes in TG2's hydrodynamic diameter. We confirmed the well-known compaction by guanosine nucleotides and extension by Ca2+, and provide evidence for TG2 conformations so far not described by structural analysis. Moreover, we analyze the influence of the peptidic Z-DON inhibitor and the R580A mutation on the conformational responsiveness of TG2 to its natural effectors. In summary, this work shows how the combination of structural and kinetic information obta...Continue Reading

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Citations

Jun 3, 2021·International Journal of Molecular Sciences·Ames C RegisterHo Young Lee
Jul 3, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Sandra RamotowskaMariusz Makowski

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