Probing the remarkable thermal kinetics of visual rhodopsin with E181Q and S186A mutants

Guo, Ying; Hendrickson, Heidi P.; Videla, Pablo E.; Chen, Ya-Na; Ho, Junming; Sekharan, Sivakumar; Batista, Víctor S.; Tully, John C.; Yan, Elsa C. Y.

doi:10.1063/1.4984818

Cited by 7 publications

(7 citation statements)

References 50 publications

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“…The obtained activation energies match well those observed for analogue dynamic processes in the α-helical region of the prokaryotic Nitrogen Regulatory Protein C (NtrC) [37] and conformational exchange near the chromophore in GFP [38]. Also the pre-exponential factors k ex ’ match values observed for dynamic processes in proteins [39]. However, the ΔG ‡ values for LZ2 C335A are about twice as high than for LZ2 C335A/E342C.…”

Section: Discussionsupporting

confidence: 78%

Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds

et al. 2019

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Nek2 is a dimeric serine/ threonine protein kinase that belongs to the family of NIMA-related kinases (Neks). Its N-terminal catalytic domain and its C-terminal regulatory region are bridged by a leucine zipper, which plays an important role in the activation of Nek2’s catalytic activity. Unusual conformational dynamics on the intermediary/slow timescale has thwarted all attempts so far to determine the structure of the Nek2 leucine zipper by means of X-ray crystallography and Nuclear Magnetic Resonance (NMR). Disulfide engineering, the strategic placement of non-native disulfide bonds into flexible regions flanking the coiled coil, was used to modulate the conformational exchange dynamics of this important dimerization domain. The resulting reduction in exchange rate leads to substantial improvements of important features in NMR spectra, such as line width, coherence transfer leakage and relaxation. These effects were comprehensively analyzed for the wild type protein, two single disulfide bond-bearing mutants and another double disulfide bonds-carrying mutant. Furthermore, exchange kinetics were measured across a wide temperature range, allowing for a detailed analysis of activation energy (ΔG‡) and maximal rate constant (k’ex). For one mutant carrying a disulfide bond at its C-terminus, a full backbone NMR assignment could be obtained for both conformers, demonstrating the benefits of the disulfide engineering. Our study demonstrates the first successful application of ‘kinetic’ disulfide bonds for the purpose of controlling the adverse effects of protein dynamics. Firstly, this provides a promising, robust platform for the full structural and functional investigation of the Nek2 leucine zipper in the future. Secondly, this work broadens the toolbox of protein engineering by disulfide bonds through the addition of a kinetic option in addition to the well-established thermodynamic uses of disulfide bonds.

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Section: Discussionsupporting

confidence: 78%

Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds

et al. 2019

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“…This value is very close to the experimentally observed barrier (22.3 kcal/mol) for the 310.5−317.65 K temperature range. 36 This value is 10.8 kcal/ mol lower than the previously reported potential energy barrier for the charge-transfer transition state (34 kcal/mol) 20 and 1.7 kcal/mol higher than the potential energy barrier calculated in the current study (21.5 kcal/mol). The resulting calculated free energy difference between Rh and bathoRh is 7.1 kcal/mol.…”

Section: Resultscontrasting

confidence: 69%

“…However, such an investigation has become important, especially, in the wake of experiments reporting an unusual kinetic behavior of the thermally induced isomerization of visual rhodopsin. 36 In conclusion, below, we show how a thermodynamic cycle method has been incorporated into the ASEC-FEG protocol 14 to compute free energy differences. Such a method is benchmarked using representative animal and microbial rhodopsins.…”

Section: Introductionmentioning

confidence: 99%

Free Energy Computation for an Isomerizing Chromophore in a Molecular Cavity via the Average Solvent Electrostatic Configuration Model: Applications in Rhodopsin and Rhodopsin-Mimicking Systems

Nikolaev

Manathunga

Orozco‐Gonzalez

et al. 2021

J. Chem. Theory Comput.

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We present a novel technique for computing the free energy differences between two chromophore "isomers" hosted in a molecular environment (a generalized solvent). Such an environment may range from a relatively rigid protein cavity to a flexible solvent environment. The technique is characterized by the application of the previously reported "average electrostatic solvent configuration" method, and it is based on the idea of using the free energy perturbation theory along with a chromophore annihilation procedure in thermodynamic cycle calculations. The method is benchmarked by computing the ground-state room-temperature relative stabilities between (i) the cis and trans isomers of prototypal animal and microbial rhodopsins and (ii) the analogue isomers of a rhodopsin-like light-driven molecular switch in methanol. Furthermore, we show that the same technology can be used to estimate the activation free energy for the thermal isomerization of systems i−ii by replacing one isomer with a transition state. The results show that the computed relative stability and isomerization barrier magnitudes for the selected systems are in line with the available experimental observation in spite of their widely diverse complexity.

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“…In this lineage, therefore, residues 186 and 181 still interact with each other—as we propose for spider Rh1—and may have an unknown function(s) other than stabilization of the PSB. In vertebrate visual pigments, Ser186 is highly conserved and interacts indirectly with Glu181 through a hydrogen-bonding network that affects many aspects of their molecular properties 13,37,38 . In these rhodopsins, a polar residue at position 186 seems to have functions other than stabilization of the PSB, indicating that the residue 186 can have diverse functions based on its close proximity to both the PSB and Glu181.…”

Section: Discussionmentioning

confidence: 99%

The counterion–retinylidene Schiff base interaction of an invertebrate rhodopsin rearranges upon light activation

et al. 2019

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Animals sense light using photosensitive proteins—rhodopsins—containing a chromophore—retinal—that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB), which requires a negatively-charged amino acid residue—counterion—for stabilization. Little is known about how the most common counterion among varied rhodopsins, Glu181, functions. Here, we demonstrate that in a spider visual rhodopsin, orthologue of mammal melanopsins relevant to circadian rhythms, the Glu181 counterion functions likely by forming a hydrogen-bonding network, where Ser186 is a key mediator of the Glu181–SB interaction. We also suggest that upon light activation, the Glu181–SB interaction rearranges while Ser186 changes its contribution. This is in contrast to how the counterion of vertebrate visual rhodopsins, Glu113, functions, which forms a salt bridge with the SB. Our results shed light on the molecular mechanisms of visible light-sensitivity relevant to invertebrate vision and vertebrate non-visual photoreception.

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Probing the remarkable thermal kinetics of visual rhodopsin with E181Q and S186A mutants

Cited by 7 publications

References 50 publications

Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds

Controlling the dynamics of the Nek2 leucine zipper by engineering of “kinetic” disulphide bonds

Free Energy Computation for an Isomerizing Chromophore in a Molecular Cavity via the Average Solvent Electrostatic Configuration Model: Applications in Rhodopsin and Rhodopsin-Mimicking Systems

The counterion–retinylidene Schiff base interaction of an invertebrate rhodopsin rearranges upon light activation

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