The binding of the small heat-shock protein αB-crystallin to fibrils of α-synuclein is driven by entropic forces

Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson’s disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

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αB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid β-peptide and β2-microglobulin

Biochemical Journal ◽

10.1042/bj20050339 ◽

2005 ◽

Vol 392 (3) ◽

pp. 573-581 ◽

Cited By ~ 104

Author(s):

Bakthisaran Raman ◽

Tadato Ban ◽

Miyo Sakai ◽

Saloni Y. Pasta ◽

Tangirala Ramakrishna ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Amyloid Fibrils ◽

Amyloid Β ◽

Small Heat Shock Protein ◽

Stable Complex ◽

Β2 Microglobulin ◽

Αb Crystallin ◽

Amorphous Aggregation ◽

Induced Aggregation

αB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of αB-crystallin on the fibril growth of the Aβ (amyloid β)-peptides Aβ-(1–40) and Aβ-(1–42). αB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Aβ-(1–40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric α-crystallins in preventing Aβ-(1–40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on α-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Aβ-(1–40). αB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Aβ-(1–42), as well as the fibril growth of Aβ-(1–40) when seeded with the Aβ-(1–42) fibril seed. Sedimentation velocity measurements show that αB-crystallin does not form a stable complex with Aβ-(1–40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. αB-crystallin binds to the amyloid fibrils of Aβ-(1–40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that αB-crystallin prevents the fibril growth of β2-microglobulin under acidic conditions. It also retards the depolymerization of β2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease.

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The small heat-shock protein αB-crystallin uses different mechanisms of chaperone action to prevent the amorphous versus fibrillar aggregation of α-lactalbumin

Biochemical Journal ◽

10.1042/bj20121187 ◽

2012 ◽

Vol 448 (3) ◽

pp. 343-352 ◽

Cited By ~ 38

Author(s):

Melissa Kulig ◽

Heath Ecroyd

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Conformational Stability ◽

Critical Role ◽

Small Heat Shock Protein ◽

Αb Crystallin ◽

Amorphous Aggregation ◽

Fibril Aggregates

Stress conditions can destabilize proteins, promoting them to unfold and adopt intermediately folded states. Partially folded protein intermediates are unstable and prone to aggregation down off-folding pathways leading to the formation of either amorphous or amyloid fibril aggregates. The sHsp (small heat-shock protein) αB-crystallin acts as a molecular chaperone to prevent both amorphous and fibrillar protein aggregation; however, the precise molecular mechanisms behind its chaperone action are incompletely understood. To investigate whether the chaperone activity of αB-crystallin is dependent upon the form of aggregation (amorphous compared with fibrillar), bovine α-lactalbumin was developed as a model target protein that could be induced to aggregate down either off-folding pathway using comparable buffer conditions. Thus when α-lactalbumin was reduced it aggregated amorphously, whereas a reduced and carboxymethylated form aggregated to form amyloid fibrils. Using this model, αB-crystallin was shown to be a more efficient chaperone against amorphously aggregating α-lactalbumin than when it aggregated to form fibrils. Moreover, αB-crystallin forms high molecular mass complexes with α-lactalbumin to prevent its amorphous aggregation, but prevents fibril formation via weak transient interactions. Thus, the conformational stability of the protein intermediate, which is a precursor to aggregation, plays a critical role in modulating the chaperone mechanism of αB-crystallin.

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