Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme.
Abstract: The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alpha-domain of the protein is formed for the whole population of molecules within 4 ms. Subsequent to this event, however, the hydrogen exchange protection kinetics are complex and highly heterogeneous. Analysis of the results by fitting to stretched exponential functions shows that a series of other intermediates is formed as a consequence of the stepwise assembly of independently nucleated local regions of structure. In some molecules the next step in folding involves the stabilisation of the remaining helix in the alpha-domain, whilst in others persistent structure begins to form in the beta-domain. The formation of native-like structure throughout the beta-domain is itself heterogeneous, involving at least three kinetically distinguishable steps. Residues in loop regions throughout the protein attain persistent structure more slowly than regions of secondary structure. There is in addition evidence for locally misfolded regions of structure that reorganise on much longer timescales. The results reveal that the native state of the protein is generated by the heterogeneous assembly of a series of locally cooperative regions of structure. This observation has many features in common with the findings of recent theoretical simulations of protein folding.
Copyright 1999 Academic Press.
Publication Date: 1999-06-17 PubMed ID: 10369782DOI: 10.1006/jmbi.1999.2741Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study examines the process of refolding equine lysozyme from guanidinium chloride, using methods like stopped flow optical experiments and hydrogen exchange pulse labelling in tandem with NMR spectroscopy. The research reveals that the restoration of the protein’s native state involves a stepwise assembly of independently formed local regions of structure, signifying a highly heterogeneous and tiered process.
Research Methodology
- The team studied the refolding process of equine lysozyme, a protein, from guanidinium chloride.
- They used various tools such as stopped flow optical methods and hydrogen exchange pulse labelling combined with NMR spectroscopy, an innovative approach that enabled them to trace almost 50 sites within the protein.
Key Findings
- One significant observation was that an extensive hydrophobic collapse happens shortly after the initiation of refolding.
- Within the first 4ms, they observed the formation of an intermediary state with persistent structures in three out of four helices within the alpha-domain of the protein.
- However, the subsequent stages of the hydrogen exchange protection kinetics proved to be complex and highly heterogeneous, indicating the formation of several other intermediates due to the sequential assembly of independently formed local structures.
- The research also found that the next step in the folding process differs across various molecules. In some, the next stages involve stabilising the remaining helix within the alpha-domain while in others, the persistent structure formation begins in the beta-domain.
- Another observation was that the formation of native-like structures throughout the beta-domain is in itself diverse, involving at least three kinetically distinguishable steps.
Implications and Conclusions
- Residues present in loop regions throughout the protein attain persistent structure at a slower pace compared to sections of secondary structure.
- Also observed were locally misfolded regions of structure which take much longer to reorganise.
- Overall the research concluded that the production of the protein’s native state involves a highly heterogeneous assembly of locally cooperative structure regions.
- This discovery aligns closely with recent theoretical simulations of protein folding and gives more insights into the stages and structural changes during protein folding.
Cite This Article
APA
Morozova-Roche LA, Jones JA, Noppe W, Dobson CM.
(1999).
Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme.
J Mol Biol, 289(4), 1055-1073.
https://doi.org/10.1006/jmbi.1999.2741 Publication
Researcher Affiliations
- Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QT, United Kingdom.
MeSH Terms
- Animals
- Circular Dichroism
- Horses
- Hydrogen
- Muramidase / chemistry
- Nuclear Magnetic Resonance, Biomolecular
- Protein Conformation
- Protein Folding
Grant Funding
- Wellcome Trust
Citations
This article has been cited 11 times.- Bhattacharjee R, Udgaonkar JB. Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein.. Protein Sci 2022 Dec;31(12):e4513.
- Kuwajima K, Yagi-Utsumi M, Yanaka S, Kato K. DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.. Molecules 2022 Jun 10;27(12).
- Pancsa R, Raimondi D, Cilia E, Vranken WF. Early Folding Events, Local Interactions, and Conservation of Protein Backbone Rigidity.. Biophys J 2016 Feb 2;110(3):572-583.
- Weber JK, Jack RL, Pande VS. Emergence of glass-like behavior in Markov state models of protein folding dynamics.. J Am Chem Soc 2013 Apr 17;135(15):5501-4.
- Maccotta A, De Pasquale C, Caruso A, Cosentino C, Alonzo G, Conte P. Reconstruction of the environmental evolution of a Sicilian saltmarsh (Italy).. Environ Sci Pollut Res Int 2013 Jul;20(7):4847-58.
- Juárez J, Taboada P, Mosquera V. Existence of different structural intermediates on the fibrillation pathway of human serum albumin.. Biophys J 2009 Mar 18;96(6):2353-70.
- Nakajima S, Kikuchi T. Analysis of the differences in the folding mechanisms of c-type lysozymes based on contact maps constructed with interresidue average distances.. J Mol Model 2007 May;13(5):587-94.
- Hamada D, Dobson CM. A kinetic study of beta-lactoglobulin amyloid fibril formation promoted by urea.. Protein Sci 2002 Oct;11(10):2417-26.
- Steinbach PJ, Ionescu R, Matthews CR. Analysis of kinetics using a hybrid maximum-entropy/nonlinear-least-squares method: application to protein folding.. Biophys J 2002 Apr;82(4):2244-55.
- Huang CY, Getahun Z, Zhu Y, Klemke JW, DeGrado WF, Gai F. Helix formation via conformation diffusion search.. Proc Natl Acad Sci U S A 2002 Mar 5;99(5):2788-93.
- Gillespie B, Plaxco KW. Nonglassy kinetics in the folding of a simple single-domain protein.. Proc Natl Acad Sci U S A 2000 Oct 24;97(22):12014-9.
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