Constitutive HSP70: oligomerization and its dependence on ATP binding

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Structure and Function

Background:

• Heat shock proteins (HSPs), particularly HSP70, play crucial roles in cellular stress response.
• The oligomeric state of proteins can significantly influence their function and interactions.
• Understanding HSP70's structural dynamics is key to elucidating its chaperone activity.

Purpose of the Study:

• To investigate the oligomeric forms of constitutive HSP70 purified from Chinese Hamster Ovary (CHO) cells.
• To determine the influence of Adenosine Triphosphate (ATP) on HSP70 oligomerization.
• To explore the binding site of ATP within HSP70 and its effect on protein assembly.

Main Methods:

• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess protein purity and molecular weight.
• Native-PAGE to identify non-denatured protein complexes and oligomeric states.
• Glutaraldehyde crosslinking to stabilize oligomeric forms for SDS-PAGE analysis.
• Autoradiography with [gamma-32P]ATP to detect ATP binding sites on HSP70.

Main Results:

• Native-PAGE revealed multiple HSP70 bands, suggesting the presence of oligomeric forms, while SDS-PAGE showed a single 70 kDa band (monomer).
• Crosslinking and SDS-PAGE identified monomer (70 kDa), dimer (153 kDa), and trimer (200 kDa) forms of HSP70.
• The proportion of oligomeric forms was modulated by ATP concentration, increasing with ATP hydrolysis and decreasing with high ATP levels (1-10 mM).
• Autoradiography confirmed that ATP binds specifically to the HSP70 monomer.

Conclusions:

• HSP70 exists in dynamic equilibrium between monomeric and oligomeric states, influenced by ATP concentration and hydrolysis.
• ATP binding to the monomeric form appears to regulate the formation and stability of HSP70 oligomers.
• Both monomeric and oligomeric HSP70 likely interact with denatured proteins during heat shock, contributing to cellular proteostasis.