Kinetically trapped structure in the renaturation of reduced oxindolealanine 62 lysozyme.
The refolded products of reduced native lysozyme and reduced OX62 lysozyme, in which Trp62 is converted to oxindolealanine (OX62) during the renaturation of sulfhydryl-disulfide interchange reactions at pH 8 and 37 degrees C, were investigated. On gel-chromatography eluted with 10% aqueous acetic acid containing 4 M urea, two peaks appeared in the refolded product of reduced OX62 lysozyme while a single peak appeared in the refolded product of reduced native lysozyme. From the analyses of the activity and primary and the tertiary structures of the derivative, the structure of the derivative from reduced native lysozyme was confirmed to be identical to that of the untreated one. On the other hand, the refolded product from reduced OX62 lysozyme had the same primary structure but a different tertiary structure compared to the untreated one. The tertiary structure of the refolded product from the reduced OX62 lysozyme was changed to that of the untreated one by the denaturation-renaturation treatment under nonreduced conditions. However, the refolded species was barely changed to that of the untreated one by incubation under physiological conditions. Therefore, the refolded product from reduced OX62 lysozyme was suggested to be a metastable and kinetically trapped product in the renaturation process of reduced OX62 lysozyme. In addition, an interaction involving the folding process of reduced lysozyme was discussed on the basis of the NMR analyses of the metastable structure.
Graduate School of Pharmaceutical Sciences and Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
T
T
Ueda
Y
Y
Abe
T
T
Ohkuri
K
K
Kawano
Y
Y
Terada
T
T
Imoto
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D004220:Disulfides; D004529:Egg White; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea
D004220:Disulfides; D010446:Peptide Fragments; C026656:oxindoleamine-62 lysozyme; D014508:Urea; D009113:Muramidase; D003488:Cyanogen Bromide