Role of the Unique Peptide Tail in Hyperthermostable Aquifex aeolicus Cochaperonin Protein 10

• Published in: Biochemistry (Easton) Vol. 44; no. 44; pp. 14385 - 14395
• Main Authors: Luke, Kathryn, Apiyo, David, Wittung-Stafshede, Pernilla
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.11.2005
• Subjects: Bacteria - chemistry; Bacteria - genetics; Chaperonin 10 - chemistry; Chaperonin 10 - genetics; Chaperonin 10 - metabolism; Chaperonin 10 - ultrastructure; Hot Temperature; Models, Molecular; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Thermodynamics
• DOI: 10.1021/bi051131l
• ISSN: 0006-2960

All known cochaperonin protein 10 (cpn10) molecules are heptamers of seven identical subunits noncovalently linked by β-strand interactions. Cpn10 from the deep-branching, hyperthermophilic bacterium Aquifex aeolicus (Aacpn10) shows high homology with mesophilic and other thermophilic cpn10 sequences, except for a 25-residue C-terminal extension not found in any other cpn10. Prior to atomic structure information, we here address the role of the tail by biophysical means. A tail-lacking variant (Aacpn10-del25) also adopts a heptameric structure in solution and exhibits nativelike substrate-refolding activity. Thermal and chemical perturbations of both Aacpn10 and Aacpn10-del25, probed by far-UV circular dichroism, demonstrate that both proteins have high thermodynamic stability. Heptamer−monomer dissociation midpoints were defined by isothermal titration calorimetry; at 25 °C, the values for Aacpn10 and Aacpn10-del25 are within 2-fold of each other and close to reported midpoints for mesophilic cpn10 proteins. In contrast, the monomer stabilities for the A. aeolicus proteins are significantly higher than those of mesophilic homologues at 30 °C; thus, heptamer thermophily is a result of more stable monomers. Electron microscopy data reveals that Aacpn10-del25 heptamers are prone to stack on top of each other forming chainlike molecules; the electrostatic surface pattern of a structural model can explain this behavior. Taken together, the unique tail in Aacpn10 is not required for heptamer structure, stability, or function; instead, it appears to be an ancient strategy to avoid cochaperonin aggregation at extreme temperatures.