Assisting the reactivation of guanidine hydrochloride-denatured aminoacylase by hydroxypropyl cyclodextrins.
Cyclodextrin is a water-soluble circular oligosaccharide with a cylinder shape characterized by exterior hydrophilic rims and an interior hydrophobic cavity, which makes it an ideal additive to prevent proteins from aggregating during refolding. In this research, three hydroxypropyl cyclodextrins (HPCDs), HP-alpha-, beta-, and gamma-CD, were used to investigate the molecular mechanism of their effects on assisting aminoacylase refolding. The aggregation and reactivation experiments suggested that at moderate concentrations, HPCDs could suppress aggregation and assist aminoacylase refolding in a concentration-dependent manner, and HP-beta-CD was the most efficient of the three HPCDs. Low concentrations of HP-alpha-CD and high concentrations of HP-gamma-CD promoted off-pathway aggregation. Spectroscopic studies indicated that the hydrophobic exposure of the unstructured species in the refolded solutions was gradually reduced by the three HPCDs with the efficiency HP-beta-CD > HP-gamma-CD > HP-alpha-CD. Furthermore, the fast phase of aminoacylase reactivation was slowed down by the addition of 75 mM HP-beta- and gamma-CD, but no significant effect was observed for HP-alpha-CD. The dissimilarity in the effects of the three HPCDs suggested that the internal cavity size played a crucial role in their antiaggregation ability. Further analysis suggested that the observations might be much more complicated than expected because of the various types of interactions between cyclodextrins and proteins in addition to their ability to bind to protein aromatic residues.
Kim
Sung-Hye
SH
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, Peoples Republic of China.
Zhang
Jun
J
Jiang
Yan
Y
Zhou
Hai-Meng
HM
Yan
Yong-Bin
YB
D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000581:Amidohydrolases / Q000737:chemistry*; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D047391:alpha-Cyclodextrins / Q000737:chemistry*; D047392:beta-Cyclodextrins / Q000737:chemistry*; D047408:gamma-Cyclodextrins / Q000737:chemistry*
D004793:Enzyme Reactivators; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; D047408:gamma-Cyclodextrins; C479658:hydroxypropyl-alpha-cyclodextrin; C479659:hydroxypropyl-gamma-cyclodextrin; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000581:Amidohydrolases; C019672:aminoacylase I; D019791:Guanidine