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Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions.

ABSTRACT: It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo-stabilization of the thermophilic rhodopsin from Thermus thermophilus JL-18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free-energy differences upon mutation. Experimental characterizations using the UV-visible spectroscopy and the differential scanning calorimetry revealed that four of ten mutations were thermo-stabilizing: V79K, T114D, A115P, and A116E. The mutation-structure relationship of the TR constructs was analyzed using molecular dynamics simulations at 300?K and at 1800?K that aimed simulating structures in the native and in the random-coil states, respectively. The native-state simulation exhibited an ion-pair formation of the stabilizing V79K mutant as it was designed, and suggested a mutation-induced structural change of the most stabilizing T114D mutant. On the other hand, the random-coil-state simulation revealed a higher structural fluctuation of the destabilizing mutant S8D when compared to the wild type, suggesting that the higher entropy in the random-coil state deteriorated the thermal stability. The present thermo-stabilization design in the extramembrane regions based on the free-energy calculation and the subsequent evaluation by the molecular dynamics may be useful to improve the production of membrane proteins for structural studies.

SUBMITTER: Akiyama T

PROVIDER: S-EPMC7894484 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions.

Akiyama Tomoki T Kunishima Naoki N Nemoto Sayaka S Kazama Kazuki K Hirose Masako M Sudo Yuki Y Matsuura Yoshinori Y Naitow Hisashi H Murata Takeshi T

Proteins 20201028 3

It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo-stabilization of the thermophilic rhodopsin from Thermus thermophilus JL-18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free-energy differences upo ...[more]

PMID: 33064333

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Project description:UnlabelledThermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog p-chlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on β-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well.ImportanceThermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into the T. thermophilus genome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

Project description:Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Dataset Information

Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions.

Publications

Further thermo-stabilization of thermophilic rhodopsin from Thermus thermophilus JL-18 through engineering in extramembrane regions.

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