NMR and Modeling Studies of Protein–Carbohydrate Interactions: Synthesis, Three‐Dimensional Structure, and Recognition Properties of a Minimum Hevein Domain with Binding Affinity for Chitooligosaccharides

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Abstract
HEV32, a 32‐residue, truncated hevein lacking eleven C‐terminal amino acids, was synthesized by solid‐phase methodology and correctly folded with three cysteine bridge pairs. The affinities of HEV32 for small chitin fragments—in the forms of N,N′,N′′‐triacetylchitotriose ((GlcNAc)3) (millimolar) and N,N′,N′′,N′′′,N′′′′,N′′′′′‐hexaacetylchitohexaose ((GlcNAc)6) (micromolar)—as measured by NMR and fluorescence methods, are comparable with those of native hevein. The HEV32 ligand‐binding process is enthalpy driven, while entropy opposes binding. The NMR structure of ligand‐bound HEV32 in aqueous solution was determined to be highly similar to the NMR structure of ligand‐bound hevein. Solvated molecular‐dynamics simulations were performed in order to monitor the changes in side‐chain conformation of the binding site of HEV32 and hevein upon interaction with ligands. The calculations suggest that the Trp21 side‐chain orientation of HEV32 in the free form differs from that in the bound state; this agrees with fluorescence and thermodynamic data. HEV32 provides a simple molecular model for studying protein–carbohydrate interactions and for understanding the physiological relevance of small native hevein domains lacking C‐terminal residues.

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