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Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation
Alternative TitleInt. J. Biochem. Cell Biol.
Gu, ZY; Zhu, X; Ni, SW; Su, Z; Zhou, HM
2004-05-01
Source PublicationINTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
ISSN1357-2725
Volume36Issue:5Pages:795-805
AbstractIt is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10 mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding. (C) 2003 Elsevier Ltd. All rights reserved.; It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10 mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding. (C) 2003 Elsevier Ltd. All rights reserved.
KeywordIntermediate Secondary Structure Aggregation Refolding Lysozyme Amyloid
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.biocel.2003.08.015
URL查看原文
Indexed BySCI
Language英语
WOS KeywordEGG-WHITE LYSOZYME ; PROTEIN DISULFIDE-ISOMERASE ; HEN LYSOZYME ; CIRCULAR-DICHROISM ; ELECTROLYTE-SOLUTIONS ; SECONDARY STRUCTURE ; ETHANOL SOLUTION ; PATHWAY ; FIBRILLOGENESIS ; EQUILIBRIUM
WOS Research AreaBiochemistry & Molecular Biology ; Cell Biology
WOS SubjectBiochemistry & Molecular Biology ; Cell Biology
WOS IDWOS:000220453900008
Citation statistics
Cited Times:66[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/4912
Collection研究所(批量导入)
Affiliation1.Tsing Hua Univ, Dept Biol Sci & Biotechnol, Beijing 100084, Peoples R China
2.Chinese Acad Sci, Inst Proc Engn, Beijing 100080, Peoples R China
Recommended Citation
GB/T 7714
Gu, ZY,Zhu, X,Ni, SW,et al. Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation[J]. INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY,2004,36(5):795-805.
APA Gu, ZY,Zhu, X,Ni, SW,Su, Z,&Zhou, HM.(2004).Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation.INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY,36(5),795-805.
MLA Gu, ZY,et al."Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation".INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 36.5(2004):795-805.
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