Molecular chaperones perform vital cellular functions under normal growth conditions and protect cells against stress-induced damage. The stress proteins Hsp70 and Hsp80 of Neurospora crassa were extracted from heat-shocked mycelium, purified to near homogeneity, and examined with respect to their oligomeric state, complex formation, and chaperoning properties. Their oligomeric state was assessed by dynamic light-scattering measurements, and both Hsp70 and Hsp80 were observed to form a range of soluble, high-molecular-mass protein aggregates. Direct interaction between Hsp70 and Hsp80 was studied by partial tryptic digestion and surface plasmon resonance (SPR). Hsp70 was immobilized on the sensor chip surface, and the binding of Hsp80 in solution was followed in real time. Proteolytic digestion revealed that Hsp70Hsp80 complex formation results in conformational changes in both proteins. The data from SPR studies yielded an equilibrium dissociation constant, KD, of 8.5 × 109 M. The chaperoning ability of Hsp70, Hsp80, and Hsp70Hsp80 was monitored in vitro by the protection of citrate synthase from thermal aggregation. The binding of nucleotides modulates the oligomeric state, chaperoning function, and hetero-oligomeric complex formation of Hsp70 and Hsp80.Key words: molecular chaperones, surface plasmon resonance, dynamic light scattering, trypsin digestion, citrate synthase, Neurospora crassa.
Study on complex formation between recombinant human thrombomodulin fragment and thrombin using surface plasmon resonance
