Abstract
Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.
Highlights
Five amino acid residues responsible for extreme stability have been identified in cytochrome c552 (HT c552) from a thermophilic bacterium, Hydrogenobacter thermophilus
The Nterminal amino acid sequence of the wild-type HT c552 protein expressed in the E. coli periplasm was determined to be AsnGlu-Gln-Leu-Ala-Lys-Gln, which was identical to that of the authentic protein purified from the native organism, H. thermophilus [3]
This indicates that the pairwise comparison with homologous cytochrome c551 (PA c551) signal peptide in the present fusion protein was correctly processed in the E. coli cells
Summary
A thermophilic hydrogen-oxidizing Gram-negative bacterium, Hydrogenobacter thermophilus, which grows optimally at 72 °C, produces a periplasmic Class I cytochrome c552 (HT c552)1 [3] This bacterial cytochrome c has greatly contributed to the understanding of protein stability through pairwise comparison with homologous cytochrome c551 (PA c551) from a mesophilic bacterium, Pseudomonas aeruginosa, which grows at 37 °C [4]. These two proteins exhibit 56% sequence identity and have almost the same backbone conformations, but HT c552 is much more stable than PA c551 [5,6,7,8,9].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
