Helix-rich transient and equilibrium intermediates of equine beta-lactoglobulin in alkaline buffer.
Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, which is similar to the helix-rich state of ELG at pH 4.0. A kinetic study was done on the folding rate of ELG and compared with bovine beta-lactoglobulin (BLG). Transient intermediates, which were observed as the burst phase of the refolding reaction, were also rich in alpha-helix. The activation enthalpy of ELG was calculated to be c.a. 80 kJ/mol, whereas that of BLG was c.a. 70 kJ/mol in the presence of 45% EGOH. The ellipticities of the transient intermediate of ELG show temperature dependence in the presence of 45% EGOH, whereas that of BLG did not show significant dependence. This study therefore extends the existence of helix-rich equilibrium and transient intermediates of predominantly beta-sheet proteins to alkaline buffer conditions.
Matsumura
Yoshitaka
Y
Department of Physics, Kansai Medical University, 18-89 Uyama-Higashi, Hirakata 573-1136, Japan.
Li
Jinsong
J
Ikeguchi
Masamichi
M
Kihara
Hiroshi
H
D000818:Animals; D002021:Buffers; D019855:Ethylene Glycol / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D020673:Protein Renaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics
D002021:Buffers; D007782:Lactoglobulins; D019855:Ethylene Glycol; D019791:Guanidine