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Molten Globule and Native State Ensemble of Helicobacter pylori Flavodoxin: Can Crowding, Osmolytes or Cofactors Stabilize the Native Conformation Relative to the Molten Globule?

Biocomputation and Complex Systems Physics Institute and Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain

Correspondence: Address reprint requests to J. Sancho, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain. E-mail: jsancho{at}unizar.es

Partly unfolded protein conformations close in energy to the native state may be involved in protein functioning and also be related to folding diseases, but yet their structure and energetics are poorly understood. One such conformation, the monomeric and well-behaved molten globule of Helicobacter pylori apoflavodoxin, is here investigated to provide, in a wide pH interval, a complete thermodynamic description of its unfolding equilibrium and the equilibrium linking molten globule and native state. All thermodynamic and molecular properties of the molten globule here analyzed are characteristic of a partly unfolded conformation, and their differences with those of the native state are typically quantitative rather than qualitative. The stability data depict a native state ensemble where the relative populations of the different intermediates are strongly modulated by pH. Whereas the molten globule is dominant at pH 2.0, at neutral pH it is just the least stable of three partly unfolded intermediates populated by this protein. It is of interest that the energy rank of these intermediates at pH 7.0 is consistent with their likelihood to overcome the native state and become the more stable conformation when the native state protein is subjected to heat or mutation stress. Given the small volume difference between molten globule and native state, neither crowding agents nor osmolytes can drive the molten globule back to the native state. This observation, which is in qualitative accord with predictions of simple excluded volume theory, indicates that molecular crowding in vivo is not an effective mechanism to minimize partial unfolding events leading to equilibrium intermediates.