Semisynthesis of a glycosylated lm7 analogue for protein folding studies

To establish a system to address questions concerning the influence of glycosylation on protein folding pathways, we have developed a semisynthetic route toward the immunity protein lm7. This four-helix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of lm7 with an Ala29Cys mutation. The semisynthetic approach relies on the solid-phase peptide synthesis (SPPS) of N-terminal thioesters (including helix I), in glycosylated or unglycosylated form, in combination with the expression of the C-terminal fragment of lm7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic lm7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this lm7 variant is not significantly altered over the unglycosylated analogue.