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. 2007 Mar 20;104(12):4846-51.
doi: 10.1073/pnas.0610630104. Epub 2007 Mar 8.

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

Thomas Durek  1 , Vladimir Yu Torbeev, Stephen B H Kent
Affiliations

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

Thomas Durek et al. Proc Natl Acad Sci U S A. .

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 (alpha)thioesters to yield a unique product. This result enables the facile preparation of a 64-residue peptide (alpha)thioester; 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 A) 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1.
Scheme 1.
Convergent synthesis of human lysozyme. The 130-aa polypeptide is assembled from four segments of comparable length in a symmetrical fashion. Key to the synthetic strategy used is the KCL of [Lys1-Trp(CHO)64]-αthioarylester and [Cys30-Trp(CHO)64]-αthioalkylester and the temporary protection of Cys65. (Inset) The 130-aa target (wild-type) sequence of human lysozyme with cysteines underlined. Steps: (i) transthioesterification; (ii) KCL; (iii) transthioesterification; (iv) NCL; (v) Cys deprotection; (vi) NCL; and (vii) Acm/CHO removal, disulfide formation, and folding. R = alkyl.
Fig. 1.
Fig. 1.
Assembly of the N-terminal half of human lysozyme by KCL. HPLC analysis of the ligation of [Lys1-Met29]-αthioarylester with [Cys30-Trp(CHO)64]-αthioalkylester in the absence of added thiol catalyst. (A) Reaction mixture at t = 1 min. (B) Reaction mixture after 90 min. (C) Reaction mixture after 5 h of ligation followed by 30 min of transthioesterification with MESNA. Chromatographic separations were performed by using a linear gradient (5–65% of buffer B in buffer A over 18 min after an initial isocratic phase of 5% buffer B in buffer A for 3 min). Buffer A: 0.1% (vol/vol) TFA in water; buffer B: 0.08% (vol/vol) TFA in acetonitrile. R, formula imageCH2COformula image(Arg)3·amide.
Fig. 2.
Fig. 2.
Assembly of the C-terminal half of human lysozyme by NCL. HPLC analysis of the ligation of [Thz65-Ala94]-αthioarylester with [Cys95-Val130]. (A) Reaction mixture shortly (1 min) after mixing of the peptide segments. (B) Reaction mixture after 2 h of ligation, followed by 6 h of methoxyamine·HCl treatment at pH 4.0. Chromatographic separations were performed by using a linear gradient (1–49% of buffer B in buffer A over 11 min after an initial isocratic phase of 1% buffer B in buffer A for 2 min). Buffer A: 0.1% (vol/vol) TFA in water; buffer B: 0.08% (vol/vol) TFA in acetonitrile.
Fig. 3.
Fig. 3.
Linking the N- and C-terminal halves to give the full-length lysozyme polypeptide. HPLC analysis of the ligation of [Lys1-Trp(CHO)64]-αMESNA thioester with [Cys65-Val130]. (A) Reaction mixture at t = 1 min. (B) Reaction mixture after 12 h of ligation. Chromatographic separations were performed as described in the Fig. 1 legend.
Fig. 4.
Fig. 4.
Characterization of synthetic human lysozyme. (A) LC analysis of purified and folded synthetic lysozyme. Chromatographic separations were performed as described in the Fig. 1 legend. (Inset) ESI-MS spectrum. The calculated mass is 14,692.7 Da. Deconvolution of the ESI-MS spectrum yields an observed mass of 14,693.4 ± 0.7 Da. (B) 2D TOCSY 1H-1H NMR spectrum showing aliphatic spin systems. (C) Clearance of a bacterial cell-wall suspension by synthetic human lysozyme (curve i). The arrowhead indicates the time when enzyme or buffer was added to the cuvette. Negative control (buffer blank, curve ii). The calculated specific enzymatic activity was 72,250 ± 1,000 units/mg of sample, comparable with recombinant human lysozyme preparations.
Fig. 5.
Fig. 5.
X-ray structure of synthetic human lysozyme. (A) Ribbon representation of the x-ray structure of synthetic human lysozyme. (B) Final 2Fo − Fc electron density map around the active site at 1.04-Å resolution. (C) Superposition of the synthetic lysozyme structure (red) with lysozyme structures obtained from biosynthetic sources (PDB ID codes: 1JSF, green; 1IWT, blue).
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