Limiting the dynamics of paramagnetic tags is crucial for the accuracy of the structural information derived from paramagnetic nuclear magnetic resonance (NMR) experiments. A hydrophilic rigid 2,2 ... Show moreLimiting the dynamics of paramagnetic tags is crucial for the accuracy of the structural information derived from paramagnetic nuclear magnetic resonance (NMR) experiments. A hydrophilic rigid 2,2 ',2 '', 2"'-( 1, 4,7, 10-tetraaz acyclo do de cane-1,4,7,10-tetrayl)tetraacetic acid (DOTA)-like lanthanoid complex was designed and synthesized following a strategy that allows the incorporation of two sets of two adjacent substituents. This resulted in a C2 symmetric hydrophilic and rigid macrocyclic ring, featuring four chiral hydroxyl-methylene substituents. NMR spectroscopy was used to investigate the conformational dynamics of the novel macrocycle upon complexation with europium and compared to DOTA and its derivatives. The twisted square antiprismatic and square antiprismatic conformers coexist, but the former is favored, which is different from DOTA. Two-dimensional 1H exchange spectroscopy shows that ring flipping of the cyclen-ring is suppressed due to the presence of the four chiral equatorial hydroxyl-methylene substituents at proximate positions. The reorientation of the pendant arms causes conformational exchange between two conformers. The reorientation of the coordination arms is slower when the ring flipping is suppressed. This indicates that these complexes are suitable scaffolds to develop rigid probes for paramagnetic NMR of proteins. Due to their hydrophilic nature, it is anticipated that they are less likely to cause protein precipitation than their more hydrophobic counterparts. Show less
Multicopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an... Show moreMulticopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an electrode. This can be overcome by designing artificial catalysts for the oxygen reduction reaction based on the active site of laccase which requires a detailed understanding of the active site. The current research is aimed at characterizing the active site of small laccase from Streptomyces coelicolor using a combination of paramagnetic NMR spectroscopy, EPR spectroscopy, mutagenesis and quantum mechanical (QM) calculations. The presence of chemical exchange at the active site of laccase attributed to the coordinating histidines is reported. QM calculations showed the importance of the orientation of the coordinating water derived ligand. Mutagenesis study showed the importance of second shell residue in stabilizing intermediates during the oxygen reduction reaction. It is also reported that by changing the pH, a new intermediate could be experimentally observed however, further research is needed to characterize this. The resonance assignment shown in the current research can be used as spies to characterize the active site of laccase. This might in future provide insight into the catalytic mechanism of oxygen reduction reaction by laccase. Show less
Dasgupta, R.; Sai Sankar Gupta, K.B.; Elam, D.; Ubbink, M.; Groot, H.J.M. de 2021
