We are exploring fundamental factors controlling electron flow in proteins and trying to apply these principles to create bio-hybrids that mimic properties of conventional semiconductors. To this end, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were expressed in E.coli in sufficient quantities and show thermal stability in temperature-dependent CD experiments comparable to wild-type protein (Tm > 90 C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins were folded correctly and formed the expected compact globular structures. Peroxidase activity assays were used to study flexibility and solvent exposure of heme binding pockets. Optical redox titrations have shown our ability to obtain reliable and reproducible data thereby allowing us to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties. Previously we have demonstrated that guanine nucleotide exchange activity of SOS for small G protein Ras is photoreversibly controlled by the peptide mimicking SOSaH helix region, which is modified with azobenzene-dimaleimide (ABDM). Recently, a novel peptide inhibitor of Ras (KRpep-2d) was reported to inhibit the guanine nucleotide exchange of Ras with GEF potently. The peptide contains two cysteine residues and the two cysteine residues are intramolecularly crosslinked by S-S bond resulting in formation of cyclic structure. The cyclic conformation is essential to exhibit inhibitory activity. In this study, we employed water soluble bifunctional azobenzene 2,2 0 -bis(sulfonate)-4,4 0 -bis(chloroacetamide)azobenzene (BSBCA22) to introduce photoswitching into SOSaH peptide and KRpep-2d. Peptides modified with BSBCA22 showed absorption spectral change accompanied by UV and visible light irradiations, reflecting cis-trans isomerization. CD spectral analysis indicated that the unmodified peptide showed random secondary structure. NMR spectra of BSBCA22-SOSaH peptide clearly revealed the two cysteine residues in the peptide intramolecularly crosslinked. CD and NMR analysis suggested BSBCA22-SOSaH peptide formed partially a-helix structure and UV and...
Ionotropic glutamate receptors are believed to undergo twisting and shortening upon activation. We used fast-scan atomic force microscopy (AFM) imaging to examine the conformational change of the GluK2 kainate receptor, integrated into a lipid bilayer, in response to activation by the agonist glutamate, either added to the imaging chamber or generated by UV photolysis of caged glutamate. In both cases, the height of the extracellular domain of the receptor fell by 0.8 nm upon activation. In contrast, there was no significant height change in response to glutamate in the presence of the GluK2 antagonist CNQX. Our study represents the first demonstration of the effect of activation on the conformation of GluK2 receptors under near-physiological conditions.
Zoospores of the aquatic fungi Blastocladiella emersonii are phototactic and a rhodopsin-guanylyl cyclase (RhGC) was identified as the putative phototaxis receptor. Due to the direct linkage between the rhodopsin and the cyclase domain, RhGC represents the first member of a novel class of enzyme linked rhodopsins. In this work, we characterized RhGC biophysically. We show that green light maximally activates RhGC and cyclic GMP is produced. After a short green laser flash, RhGC (D525) converts in 8 ms into a blue-shifted signaling state P380 and recovers within 100 ms. RhGC expresses well in Xenopus oocytes, yeast, CHO cells, mammalian neurons and cyclic GMP production was light dose-dependent, rapid and reproducible. Thus, RhGC is a versatile tool for optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.