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Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments.

ABSTRACT: Recent theoretical and experimental results point to the existence of small barriers to protein folding. These barriers can even be absent altogether, resulting in a continuous folding transition (i.e., downhill folding). With small barriers, the detailed properties of folding ensembles may become accessible to equilibrium experiments. However, further progress is hampered because folding experiments are interpreted with chemical models (e.g., the two-state model), which assume the existence of well defined macrostates separated by arbitrarily high barriers. Here we introduce a phenomenological model based on the classical Landau theory for critical transitions. In this physical model the height of the thermodynamic free energy barrier and the general properties of the folding ensemble are directly obtained from the experimental data. From the analysis of differential scanning calorimetry data alone, our model identifies the presence of a significant (>35 kJ/mol) barrier for the two-state protein thioredoxin and the absence of a barrier for BBL, a previously characterized downhill folding protein. These results illustrate the potential of our approach for extracting the general features of protein ensembles from equilibrium folding experiments.

SUBMITTER: Munoz V

PROVIDER: S-EPMC539728 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments.

Muñoz Victor V Sanchez-Ruiz Jose M JM

Proceedings of the National Academy of Sciences of the United States of America 20041209 51

Recent theoretical and experimental results point to the existence of small barriers to protein folding. These barriers can even be absent altogether, resulting in a continuous folding transition (i.e., downhill folding). With small barriers, the detailed properties of folding ensembles may become accessible to equilibrium experiments. However, further progress is hampered because folding experiments are interpreted with chemical models (e.g., the two-state model), which assume the existence of ...[more]

PMID: 15591110

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Project description:Earliest events in the aggregation process, such as single molecule reconfiguration, are extremely important and the most difficult to characterize in experiments. To this end, we have used well-tempered bias exchange metadynamics simulations to determine the equilibrium ensembles of an insulin molecule under amyloidogenic conditions of low pH and high temperature. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodynamic and kinetic model of insulin folding. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in solution, namely, the R-, the Rf-, and the T-states of insulin. The lowest free-energy structure had a root mean square deviation of only 0.15 nm from native x-ray structure. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three additional long-lived, metastable states from the bin-based model. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also determined the lowest free-energy path between two longest-lived metastable states and confirm earlier findings of non-two-state folding for insulin through a folding intermediate. The ensemble containing the monomeric intermediate retained 58% of native hydrophobic contacts, however, accompanied by a complete loss of native secondary structure. We have discussed the relative importance of nativelike versus nonnative tertiary contacts for the folding transition. We also provide a simple measure to determine the importance of an individual residue for folding transition. Finally, we have compared and contrasted this intermediate with experimental data obtained in spectroscopic, crystallographic, and calorimetric measurements during early stages of insulin aggregation. We have also determined stability of monomeric insulin by incubation at a very low concentration to isolate protein-protein interaction effects.

Dataset Information

Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments.

Publications

Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments.

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