The unfolding mechanism and the disulfide structures of denatured lysozyme.
The mechanism of denaturation and unfolding of lysozyme has been characterized here using the method of disulfide scrambling. Under denaturing conditions (urea, guanidinium hydrochloride (GdmCl), guanidinium thiocyanate (GdmSCN), or elevated temperature) and in the presence of thiol initiator, lysozyme denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers. To denature 50% of the native lysozyme requires 1.1 M of GdmSCN, 2.8 M of GdmCl and 7.4 M of urea, respectively. High temperature (75 degrees C) denatures the native lysozyme quantitatively within 20 min. Analysis by reversed-phase high-performance liquid chromatography reveals that urea and GdmCl denatured lysozyme comprise a single predominant disulfide isomer, designated as X-lysozyme-a, regardless of the concentration of the denaturant. X-Lysozyme-a was shown to adopt the beads-form structure with its four disulfide bonds formed by four consecutive pairs of cysteines (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127). The conspicuous absence of partially structured unfolding intermediates of lysozyme contrasts to that found in the case of alpha-lactalbumin and accounts for the widely observed two-stage mechanism of lysozyme unfolding.
Chang
Jui-Yoa
JY
The Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, USA. rowen.chang@uth.tmc.edu
Li
Li
L
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007536:Isomerism; D008623:Mercaptoethanol / Q000494:pharmacology; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D004220:Disulfides; D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate; D008623:Mercaptoethanol; D014508:Urea; D009113:Muramidase; D019791:Guanidine