Physicochemical Properties of the Mammalian Molecular Chaperone HSP60

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle

Rapid Electrophoretic Staining and Destaining of Polyacrylamide Gels

Coomassie brilliant blue (CBB) dyes have been commonly used for the staining of protein bands in polyacrylamide gel electrophoresis (PAGE) gels. However, the staining and destaining of CBB dyes are time-consuming, and the use of methanol is hazardous to one’s health. I introduce a rapid electrophoretic destaining method using a semi-dry transfer unit and a high current power supply. In this method, ethanol was used instead of the hazardous methanol. Most of the protein bands became visible in 30 min. After a secondary destaining step, residual CBB was completely destained. The detection limit for a tested protein (5 ng) was higher than that of the conventional method. Therefore, this method is superior in its speed, safety, low cost, and sensitivity

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

Protein folding aided by the GroEL/GroES chaperonin has been assumed to involve free substrate polypeptide enclosed in a chaperonin cage. This work implies that substrates in fact bind GroEL and peek out from the cage, with their fate depending on whether they subsequently topple to its inside or outside

Hydrophilic Residues at the Apical Domain of GroEL Contribute to GroES Binding but Attenuate Polypeptide Binding

The GroES binding site at the apical domain of GroEL, mostly consisting of hydrophobic residues, overlaps largely with the substrate polypeptide binding site. Essential contribution of hydrophobic interaction to the binding of both GroES and polypeptide was exemplified by the mutant GroEL(L237Q) which lost the ability to bind either of them. The binding site, however, contains three hydrophilic residues, E238, T261, and N265. For GroES binding, N265 is essential since GroEL(N265A) is unable to bind GroES. E238 contributes to rapid GroES binding to GroEL because GroEL(E238A) is extremely sluggish in GroES binding. Polypeptide binding was not impaired by any mutations of E238A, T261A, and N265A. Rather, these mutants, especially GroEL(N265A), showed stronger polypeptide binding affinity than wild-type GroEL. Thus, these hydrophilic residues have a dual role; they help GroES binding on one hand but attenuate polypeptide binding on the other hand