5 research outputs found
QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins
Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity
The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols
Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying sigma antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.11Nsciescopu
Author Correction: QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins
QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins
Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity
Probing Factor Xa ProteināLigand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands
The accurate prediction of proteināligand binding
affinity
belongs to one of the central goals in computer-based drug design.
Molecular dynamics (MD)-based free energy calculations have become
increasingly popular in this respect due to their accuracy and solid
theoretical basis. Here, we present a combined study which encompasses
experimental and computational studies on two series of factor Xa
ligands, which enclose a broad chemical space including large modifications
of the central scaffold. Using this integrated approach, we identified
several new ligands with different heterocyclic scaffolds different
from the previously identified indole-2-carboxamides that show superior
or similar affinity. Furthermore, the so far underexplored terminal
alkyne moiety proved to be a suitable non-classical bioisosteric replacement
for the higher halogenāĻ aryl interactions. With this
challenging example, we demonstrated the ability of the MD-based non-equilibrium
free energy calculation approach for guiding crucial modifications
in the lead optimization process, such as scaffold replacement and
single-site modifications at molecular interaction hot spots