¹⁹F-NMR Detect Beta-arrestin1 Conformational Changes

IntroductionWith the development in biological science and technology, we are having deeper insight into the molecular mechanisms underlying the function of biomacromolecule complex. Conformational changes play important roles in intra-and intermolecular interaction and mediate the function of many proteins. Conformational changes in proteins include swing of individual amino acid residue or the whole functional domain. This process varies from picosecond to seconds, time that many intermediate transition states exist, which involving in protein function or ligand binding. X-ray diffraction or electron microscopy (EM) can hardly capture these states, obstructing revealing the details of dynamic changes. Meanwhile, sample preparation of biomolecular complex suitable for crystallographic or EM study faces difficulties, with time-consuming crystallization or complicated process respectively. As an alternative method, nuclear magnetic resonance (NMR) outstands to detect protein conformational changes nearly physiological condition in certain time scales. 100% naturally abundance and a spin 1/2 of 19F atom give 19F-NMR increasing popularity. To add to this,19F is absent from all naturally occurring biomacromolecules, high gyromagnetic ratio that results in excellent sensitivity to surrounding chemical environment, a wide range of chemical shift, so you can see the structure of biological macromolecules more subtle change, and dynamic changes. Moreover, whereas most NMR signal suffered from line broadening when they are utilized to study the dynamic conformational change of big bio-molecular complex, the sensitivity of the 19F-NMR allowed researcher to observe the detial of conformational change due to its high sensitivity. Fluorescence is a widely used molecular orientation and conformational change detection, fluorescence quenching technique be observed from the changes in the two molecules of approximately 10nm.Objectives1. Synthesis of a sensitive fluorine probe to detect conformational change of proteins.2 Examining the conformational changes during beta-arrestin1 activationMethodsUse a tyrosine phenol lyase to synthesis a tyrosine analogue F2Y. The Gene codon expansion and metabolic insertion technologies are used to incorporate the un-naturalamino acid F2Y into protein in a prokaryotic expression systemResult1 We synthesized difluorotyrosine, and developed a system to insert this difluorotyrosine into the beta-arrestinl protein.2, The 19F labeled beta-arrestinl protein has 100% incorporation rate of the F2Y- unnatural aminoacid and developed sensitive NMR signals to study the conformational changes during arrestin activation. 3, Using labeled 19F-F2Y-beta-arrestinl, we have observed conformational changes of the C-terminal tail of beta-arrestinl during its activation.Conclusion1, difluorotyrosine is a sensitive probe to detect structural changes of proteins. We have developed a system that have high efficiency to incorporate the F2Y into the beta-arrestinl to study the GPCR signaling 2, Beta-arrestinl C-terminal tail is buried in its inactive state.. During the activation, the C-terminal of beta-arrestinl is substantially exposed to the solution.