Mechanism of Chaperone Function in Small Heat Shock Proteins

Abstract

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. 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 ␣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. Our results suggest that the states recognized by ␣A-crystallin are non-native excited states distinct from the unfolded state. Stable complexes are formed when the free energy of binding to ␣A-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 ␣A-crystallin oligomeric structure.In the crowded molecular environment of the cell, protein folding, stability, and solubility are critically dependent on a specialized protein machinery consisting of multiple superfamilies of heat shock proteins, the molecular chaperones. By recognizing and binding non-native protein states, chaperones protect the cell from the toxic and pathogenic consequences of protein aggregation (1, 2). These non-native states are not only a byproduct of the folding process but are also populated through fluctuations in the native structure, the rate of which increases under extreme physicochemical conditions. The diversity of the molecular chaperones has been associated with distinct roles at different stages of the protein life cycle and in response to different cellular conditions (3).The small heat shock protein (sHSP) 1 superfamily consists of oligomeric proteins of 4 -40 subunits with a molecular mass of less than 40 kDa/subunit (4 -6). The exact role of the sHSP in the heat shock response is poorly understood, although they have been associated with a spectrum of functions under permissive temperatures (7-10). In humans, six sHSP have been identified in various tissues (6). The ␣-crystallins are the main protein component in the lens, where they play a critical role in establishing and maintaining its optical properties (11-13). It has been hypothesized that ␣-crystallin chaperone function prevents the early aggregation and precipitation of lens proteins. In non-l...

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

“…The effect is not only seen with the highly purified recombinant preparation of ␣A-and ␣B-crystallin but also with bovine ␣ Lcrystallin (Table I). The weak binding can be inferred from the fact that the binding is reversible at least partly, and the binding affinity is low compared with that, for example, between ␣B-crystallin and the destabilized T4 lysozymes that are in the low submicromolar range (40). Here both chaperone and substrate, involved in the binding interaction, exist in states not far from the native states of the proteins.…”

“…Native state fluctuations can give rise to "ladder states," having small energy differences centering around the ground state energy (41,42). In a series of papers (40,(43)(44)(45)) from Mchaourab and co-workers, it has been demonstrated that ␣-crystallin can efficiently recognize and bind these degenerate native-like states of the substrates with varying affinity. From thermodynamic considerations, they have shown that the minor alteration in the stabilization energy of a substrate can trigger binding with the chaperone (44,45).…”

Differential recognition of natural and nonnatural substrate by molecular chaperone α‐crystallin—A subunit exchange study

“…But, when a nonnatural substrate carbonic anhydrase remained bound to the chaperone, further loss in subunit exchange rate was Differential Recognition of Natural and Nonnatural Substrate by Molecular Chaperone -Crystallin-A Subunit Exchange Study -Crystallin prevents the aggregation of a large variety of substrates, e.g., insulin, -lactalbumin, lysozyme, conalbumin, alcohol dehydrogenase, citrate synthase, xylose reductase, etc. [9][10][11][12][13][14][15][16][17][18][19] These substrates include proteins, which are of low molecular weight [e.g., insulin (6 kDa)], comparable to -crystallin subunit molecular weight [e.g., carbonic anhydrase (29 kDa)] and high molecular weight [e.g., alcohol dehydragenase (150 kDa)]. Besides, it recognizes substrates under various stress conditions, such as heat, disulphide cleavage, UV light exposure, oxidative stress, etc.…”

“…Besides, it recognizes substrates under various stress conditions, such as heat, disulphide cleavage, UV light exposure, oxidative stress, etc. [9][10][11][12][13][14][15][16][18][19][20][21] These in vitro substrates belong to no particular category in terms of sequence or threedimensional structure. In addition to these ''nonnatural'' substrates, -crystallin prevents the aggregation of a number of its own substrates, which are present in the lens.…”

Mechanism of Chaperone Function in Small Heat-shock Proteins

“…In a recent study, we reported the equilibrium binding of ␣A-crystallin to destabilized mutants of T4 lysozyme (24). The advantage of these mutants is that they do not aggregate on the time scale of binding and their equilibrium folding constants are in the 10 4 -10 7 range.…”