Polyvinylpyrrolidone 40 assists the refolding of bovine carbonic anhydrase B by accelerating the refolding of the first molten globule intermediate.
Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I(1)) to the second one (I(2)) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I(1), resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I(1). These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I(1) and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I(1) does not block the aggregation pathway directly but decreases the energy barrier for I(1) to refold to I(2) and thus reduces the accumulation of I(1). These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.
Jiang
Yan
Y
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Yan
Yong-Bin
YB
Zhou
Hai-Meng
HM
D000818:Animals; D024401:Carbonic Anhydrase I / Q000378:metabolism*; D002417:Cattle; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D018832:Molecular Chaperones; D010592:Pharmaceutic Aids / Q000494:pharmacology*; D011205:Povidone / Q000494:pharmacology*; D017510:Protein Folding
D018832:Molecular Chaperones; D010592:Pharmaceutic Aids; D024401:Carbonic Anhydrase I; D011205:Povidone; D019791:Guanidine