Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k/K values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.
Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.
Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.
iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin’s C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin’s N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4–8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.
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