Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability

Yasuda, Satoshi; Hayashi, Tomohiko; Kajiwara, Yuta; Murata, Takeshi; Kinoshita, Masahiro

doi:10.1063/1.5082217

Cited by 10 publications

(60 citation statements)

References 60 publications

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“…The EC and IC portions in the folded state, which consist of significantly many hydrophilic residues, should remain exposed to water even in the denatured state. That is, the folding process of the TM portion occurs in nonpolar environment while that of EC and IC portions occurs in aqueous environment, which was rationalized in our earlier work (Yasuda et al 2019). We assume that the TM portion in the denatured state possesses neither the effective packing of side chains nor the intramolecular hydrogen bonding.…”

Section: Outline Of Our Theoretical Methodsmentioning

confidence: 79%

“…Taken together, we can employ a simplified model for the bulk solvent mentioned above: an ensemble of neutral hard spheres whose diameter and packing fraction are set at those of water at ambient temperature and pressure. Refer to our earlier publications for more details (Kajiwara et al 2016;Kajiwara et al 2017;Yasuda et al 2016;Yasuda et al 2017;Yasuda et al 2019).…”

Section: Solvent For a Membrane Proteinmentioning

confidence: 99%

“…The PMF is weaker and shorter ranged than the direct interaction energy. We optimized E NP 5k B T 0 for membrane proteins (Kajiwara et al 2018;Yasuda et al 2017;Yasuda et al 2019). The PMF is a linear, short-ranged function of the distance.…”

Section: Energetic Componentmentioning

confidence: 99%

“…Thus, ΔF/(k B T 0 N r ) evaluated at a temperature T = T 0 is a good measure of the thermostability. The lower the measure, the higher the thermostability (k B T 0 N r ) = ΔU/(k B T 0 N r )−ΔS/(k B N r ) can be regarded as a measure of the thermostability (Yasuda et al 2019). The lower the measure is, the higher the thermostability is.…”

Section: Physical Picture Of Thermal Denaturationmentioning

confidence: 99%

“…Therefore, the free-energy change upon protein folding is reformulated to consider the EC and IC portions. Refer to our recent publication for more details (Yasuda et al 2019).…”

Section: Extension To Microbial Rhodopsinsmentioning

confidence: 99%

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Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

et al. 2020

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Thermostabilization of a membrane proteins, especially G-protein-coupled receptors (GPCRs), is often necessary for biochemical applications and pharmaceutical studies involving structure-based drug design. Here we review our theoretical, physicsbased method for identifying thermostabilizing amino acid mutations. Its novel aspects are the following: The entropic effect originating from the translational displacement of hydrocarbon groups within the lipid bilayer is treated as a pivotal factor; a reliable measure of thermostability is introduced and a mutation which enlarges the measure to a significant extent is chosen; and all the possible mutations can be examined with moderate computational effort. It was shown that mutating the residue at a position of N BW = 3.39 (N BW is the Ballesteros-Weinstein number) to Arg or Lys leads to the stabilization of significantly many different GPCRs of class A in the inactive state. Up to now, we have been successful in stabilizing several GPCRs and newly solving three-dimensional structures for the muscarinic acetylcholine receptor 2 (M2R), prostaglandin E receptor 4 (EP4), and serotonin 2A receptor (5-HT 2A R) using X-ray crystallography. The subjects to be pursued in future studies are also discussed.

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Section: Outline Of Our Theoretical Methodsmentioning

confidence: 79%

Section: Solvent For a Membrane Proteinmentioning

confidence: 99%

Section: Energetic Componentmentioning

confidence: 99%

Section: Physical Picture Of Thermal Denaturationmentioning

confidence: 99%

“…Therefore, the free-energy change upon protein folding is reformulated to consider the EC and IC portions. Refer to our recent publication for more details (Yasuda et al 2019).…”

Section: Extension To Microbial Rhodopsinsmentioning

confidence: 99%

See 3 more Smart Citations

Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

et al. 2020

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A methodology for creating thermostabilized mutants of G‐protein coupled receptors by combining statistical thermodynamics and evolutionary molecular engineering

et al. 2022

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We constructed a methodology for thermostabilizing a G‐protein coupled receptor (GPCR) in the inactive state whose wild‐type (WT) structure is unknown solely by multiple amino‐acid mutations without the ligand binding. It is a combination of our recently developed theory based on statistical thermodynamics and site‐directed saturation mutagenesis, a method often employed in evolutionary molecular engineering. First, the WT structure is predicted using the homology modeling. Second, a key residue is determined by our statistical‐thermodynamics theory using suitably modeled mutant structures. Many of 19 different single mutations for the key residue are expected to produce significantly higher stabilization. Third, we undertake to mutate not only the key residue but also a few more residues whose side chains are close to the side chain of the key residue. The whole mutational space is then efficiently explored by introducing site‐directed saturation mutations, and a gene (mutant) library is constructed using the small‐intelligent and fully automatic single‐tube recombination methods. Each mutant is expressed in Escherichia coli cells, and highly stabilized mutants are sorted out using a fluorescence‐screening technique. The methodology was illustrated for the serotonin 2A receptor, 5‐HT2AR, for stabilizing its inactive state. We could identify a double mutant whose apparent midpoint temperature of thermal denaturation is higher than that of a thermostabilized double mutant previously reported by ~8.9°C and that of the WT by over 15°C. Moreover, it exhibits higher binding affinity for spiperone, an antagonist which was previously proved to stabilize 5‐HT2AR in the inactive state.

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A methodology for creating mutants of G‐protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering

et al. 2022

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We challenged the stabilization of a G‐protein coupled receptor (GPCR) in the active state solely by multiple amino‐acid mutations without the agonist binding. For many GPCRs, the free energy of the active state is higher than that of the inactive state. When the inactive state is stabilized through the lowering of its free energy, the apparent midpoint temperature of thermal denaturation Tm exhibits a significant increase. However, this is not always the case for the stabilization of the active state. We constructed a modified version of our methodology combining statistical thermodynamics and evolutionary molecular engineering, which was recently developed for the inactive state. First, several residues to be mutated are determined using our statistical‐thermodynamics theory. Second, a gene (mutant) library is constructed using Escherichia coli cells to efficiently explore most of the mutational space. Third, for the mutant screening, the mutants prepared in accordance with the library are expressed in engineered Saccharomyces cerevisiae YB14 cells which can grow only when a GPCR mutant stabilized in the active state has signaling function. For the adenosine A2A receptor tested, the methodology enabled us to sort out two triple mutants and a double mutant. It was experimentally corroborated that all the mutants exhibit much higher binding affinity for G protein than the wild type. Analyses indicated that the mutations make the structural characteristics shift toward those of the active state. However, only slight increases in Tm resulted from the mutations, suggesting the unsuitability of Tm to the stability measure for the active state.

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Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability

Cited by 10 publications

References 60 publications

Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

A methodology for creating thermostabilized mutants of G‐protein coupled receptors by combining statistical thermodynamics and evolutionary molecular engineering

A methodology for creating mutants of G‐protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering

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