Semisynthesis of a glycosylated Im7 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 Im7. This fourhelix protein has been used extensively as model protein for folding studies. Native chemical ligation (NCL) affords an N-linked chitobiose glycoprotein analogue of Im7 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 Im7 (containing helices II-IV). Detailed kinetic and thermodynamic analysis of the protein folding behavior reveals that semisynthetic Im7 analogues are well suited for protein folding studies and that the folding mechanism of the glycoprotein of this Im7 variant is not significantly altered over the unglycosylated analogue.
Hackenberger
Christian P R
CP
Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Friel
Claire T
CT
Radford
Sheena E
SE
Imperiali
Barbara
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000138:chemical synthesis*; D003087:Colicins / Q000138:chemical synthesis*; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics
D001426:Bacterial Proteins; D003087:Colicins; C007104:colicin immunity proteins