Macromolecular crowding extended to a heptameric system: the co-chaperonin protein 10

Macromolecular crowding extended to a heptameric system : the co-chaperonin protein 10

Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.

Weise, Christoph,1973-Umeå universitet,Kemiska institutionen(Swepub:umu)chwe0023 (author)
Sparrman, Tobias,1970-Umeå universitet,Kemiska institutionen(Swepub:umu)tosp0001 (author)
Wolf-Watz, Magnus,1971-Umeå universitet,Kemiska institutionen(Swepub:umu)maswoz04 (author)
Wittung-Stafshede, Pernilla,1968-Umeå universitet,Kemiska institutionen(Swepub:umu)pewi0014 (author)
Umeå universitetKemiska institutionen (creator_code:org_t)

In:Biochemistry: American Chemical Society (ACS)50:14, s. 3034-30440006-29601520-4995

By the author/editor: Aguilar, Ximena, ...; Weise, Christoph ...; Sparrman, Tobias ...; Wolf-Watz, Magnu ...; Wittung-Stafshed ...

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