Nocturnal bruxism may be secondary to nocturnal gastroesophageal reflux, occurring via sleep arousal and often together with swallowing. The physiologic link between bruxism and the increase in salivation needs to be investigated.
Mesophilic cytochrome c 551 of Pseudomonas aeruginosa (PA c 551 ) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c 552 (HT c 552 ), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c 552 through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c 551 could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c 552 , there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.Heat-stable proteins from thermophilic bacteria usually exhibit main chain foldings similar to those of mesophilic counterparts. Mutational studies on mesophilic proteins modeled with respect to thermophilic counterparts have proved that specific side chain interactions in the thermophiles are partially responsible for the higher stability (1-3). Thermodynamic analysis has also indicated that a thermophilic protein can be stabilized through global interaction throughout the molecule (4). It remains enigmatic as to how many amino acid substitutions contribute to the stability of a natural thermophilic protein (5). In some cases, a mesophilic protein only acquires the stability of the thermophilic counterpart after substantial exchanges of a linear sequence; groups of individual mutations are not sufficient (6). Multiple mutations in mesophilic proteins that completely increase the stability to the levels of thermophilic counterparts would provide important information about relationships between local side chain interactions and overall protein stability and demonstrate that the thermophilic character can depend on a limited number of strong noncovalent interactions.Cytochrome c is a powerful tool for characterizing protein stability because structural information on a variety of cytochromes c is available, and heterologous expression systems for holopoteins have been established (7). Cytochrome c 551 (PA c 551 )1 from a mesophile, Pseudomonas aeruginosa, and cytochrome c 552 (HT c 552 ) from a thermophile, Hydrogenobacter thermophilus, are 82-and 80-amino acid proteins, respectively, each with a covalently attached heme. These proteins exhibit 56% sequence identity (8) and almost the same main chain foldings (9), but HT c 552 exhibits much higher stability compared with PA c 551 (10). On a structural comparison between HT c 552 (9) and PA c 551 (11), we identified three distal regions responsible for the higher stability of the former (9). The single mutation Val-78 to Ile (V78I), and two double mutations Phe-7 to Ala/Val-13...
Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.
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