Abstract
To elucidate the mechanism of alphaA-crystallin chaperone function, a detailed thermodynamic analysis of its binding to destabilized, site-directed mutants of T4 lysozyme was carried out. The selected mutants form a ladder of stabilities spanning the 5-10 kcal/mol range of free energy of unfolding. The crystal structures of the majority of the mutants have been previously determined and found to be similar to that of the wild type with no evidence of static local unfolding. Complex formation between alphaA-crystallin and T4 lysozyme was observed directly via the changes in the electron paramagnetic resonance lineshape of a nitroxide introduced at a non-destabilizing, solvent exposed site in T4 lysozyme. AlphaA-crystallin differentially interacts with the mutants, binding the more destabilized ones to a larger extent despite the similar structure of their native states. Our results suggest that the states recognized by alphaA-crystallin are non-native excited states distinct from the unfolded state. Stable complexes are formed when the free energy of binding to alphaA-crystallin is on the order of the free energy associated with the transition from the excited state to the native state. Biphasic binding isotherms reveal two modes of interactions with distinct affinities and stoichiometries. Highly destabilized mutants preferentially bind to the high capacity mode, suggesting conformational preference in the use of each mode. Furthermore, binding can be enhanced by increased temperature and pH, which may be reflecting conformational changes in alphaA-crystallin oligomeric structure.
Highlights
To elucidate the mechanism of ␣A-crystallin chaperone function, a detailed thermodynamic analysis of its binding to destabilized, site-directed mutants of T4 lysozyme was carried out
Complex formation between ␣A-crystallin and T4 lysozyme was observed directly via the changes in the electron paramagnetic resonance lineshape of a nitroxide introduced at a non-destabilizing, solvent exposed site in T4 lysozyme. ␣A-Crystallin differentially interacts with the mutants, binding the more destabilized ones to a larger extent despite the similar structure of their native states
One of the mechanisms of protein aggregation involves the increase in the equilibrium population of non-native states, including the globally unfolded state, characterized by exposed hydrophobic surfaces
Summary
SHSP should recognize and bind proteins prior to nucleation of aggregation, i.e. should detect the increased excursion of destabilized proteins toward the aggregation-prone non-native states This implies that binding can be induced by a progressive reduction in the folding equilibrium constant of a particular substrate until a stable complex is formed. The free energy of folding of the mutants can be characterized as a function of physicochemical variables such as pH and temperature and correlated with changes in the binding to the chaperone This study uses this approach to gain insight into the mechanism of ␣A-crystallin chaperone function using T4 lysozyme as a substrate. The two modes have distinct energetic thresholds for binding and may be associated with the recognition of different conformational states of T4L These results are discussed in the context of the coupling between the dynamics of the oligomeric structure and the chaperone function in sHSP. The knowledge of the structure and folding of the T4L variants provides new insight into the recognition and binding events and the energetics associated with them
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