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
Sun, J.; Chikunova, A.; Boyle, A.L.; Voskamp, P.; Timmer, M.; Ubbink, M. 2023
Evolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non-catalytic residues, 11%... Show moreEvolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non-catalytic residues, 11% of all residues, in class A beta-lactamase were analyzed by studying the effect of 146 mutations on in cell and in vitro activity, folding, structure, and stability. Residues around the catalytic residues (second shell) contribute to fine-tuning of the active site structure. Mutations affect the structure over the entire active site and can result in stable but inactive protein. Conserved residues farther away (third shell) ensure a favorable balance of folding versus aggregation or stabilize the folded form over the unfolded state. Once folded, the mutant enzymes are stable and active and show only localized structural effects. These residues are found in clusters, stapling secondary structure elements. The results give an integral picture of the different roles of essential residues in enzymes. Show less
Miao, Q.; Nitsche, C.; Orton, H.; Overhand, M.; Otting, G.; Ubbink, M. 2022
Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great... Show moreParamagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen. Show less
Skinner, S.P.; Follmer, A.H.; Ubbink, M.; Poulos, T.L.; Houwing-Duistermaat, J.J.; Paci, E. 2021
Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of... Show moreOptimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit. Show less
Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific... Show moreGlycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts. Show less
Dasgupta, R.; Sai Sankar Gupta, K.B.; Groot, H.J.M. de; Ubbink, M. 2021
The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, one of the most abundant renewable biomaterials in nature. We demonstrate that... Show moreThe single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, attributed to an enzyme induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction. Show less
Son, M. van; Schilder, J.T.; Di Savino, A.; Blok, A.J.; Ubbink, M.; Huber, M.I. 2020
