Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme

Durek, Thomas, Torbeev, Vladimir Yu and Kent, Stephen B. H. (2007) Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme. Proceedings of the National Academy of Sciences of the United States of America, 104 2: 4846-4851. doi:10.1073/pnas.0610630104


Author Durek, Thomas
Torbeev, Vladimir Yu
Kent, Stephen B. H.
Title Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme
Journal name Proceedings of the National Academy of Sciences of the United States of America   Check publisher's open access policy
ISSN 0027-8424
1091-6490
Publication date 2007-03-20
Sub-type Article (original research)
DOI 10.1073/pnas.0610630104
Open Access Status Not Open Access
Volume 104
Issue 2
Start page 4846
End page 4851
Total pages 6
Place of publication Washington, D.C
Publisher National Academy of Sciences of the U.S.
Language eng
Subject 030406 Proteins and Peptides
Abstract In this article, we report the total chemical synthesis of human lysozyme. Lysozyme serves as a widespread model system in various fields of biochemical research, including protein folding, enzyme catalysis, and amyloidogenesis. The 130-aa wild-type polypeptide chain of the human enzyme was assembled from four polypeptide segments by using native chemical ligation in a fully convergent fashion. Key to the assembly strategy is the application of the recently developed kinetically controlled ligation methodology, which provides efficient control over the ligation of two peptide αthioesters to yield a unique product. This result enables the facile preparation of a 64-residue peptide αthioester; this segment is joined by native chemical ligation to a 66-aa Cys peptide, to yield the target 130-aa polypeptide chain. The synthetic polypeptide chain was folded in vitro into a defined tertiary structure with concomitant formation of four disulfides, as shown by 2D TOCSY NMR spectroscopy. The structure of the synthetic human lysozyme was confirmed by high-resolution x-ray diffraction, giving the highest-resolution structure (1.04 Å) observed to date for this enzyme. Synthetic lysozyme was obtained in good yield and excellent purity and had full enzymatic activity. This facile and efficient convergent synthesis scheme will enable preparation of unique chemical analogs of the lysozyme molecule and will prove useful in numerous areas of lysozyme research in the future.
Keyword chemical protein synthesis
kinetically controlled ligation
native chemical ligation
peptide thioester
protein folding
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Institute for Molecular Bioscience - Publications
 
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