Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions.
The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.
Sasahara
Kenji
K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, Japan. Kenjisa@intra.niddk.nih.gov
Demura
Makoto
M
Nitta
Katsutoshi
K
D000143:Acids / Q000494:pharmacology; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D005260:Female; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics
D000143:Acids; D007768:Lactalbumin; C421864:hen egg lysozyme; D009113:Muramidase; D019791:Guanidine