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Combination of OPSY and PhD-PHIP results in enhanced sensitivity in PHIP
摘要： Despite the large degree of polarization in PHIP experiments compared to the Boltzmann factor, the presence of a large amount of non-reacted molecules with thermal polarization is an important obstacle when dealing with very diluted samples. The feasibility of enhancing both sensitivity and resolution in a single experiment by combining two well established pulse sequences, OPSY and PHD-PHIP is presented. OPSY is used as a block for filtering the signals originated from thermally polarized protons. PhD-PHIP, on the other hand, is used as an acquisition block, increasing the resolution and further improving the sensitivity by preventing signal canceling in the presence of magnetic field inhomogeneities. Experiments in a complex sample with very low hyperpolarization levels are presented showing the excellent performance of the method.
关键词： Echo trains,PhD-PHIP,Spin dynamics,CPMG,J-spectroscopy,Hyperpolarization,Pulse sequences,Parahydrogen,SABRE,J-coupling,NMR,OPSY,PHIP
更新于2025-09-23 15:23:52
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Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser
摘要： Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s?1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s?1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s?1. The μs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.
关键词： Adenine riboswitch,B-factor,CPMG,Aptamer,Conformational exchange
更新于2025-09-16 10:30:52