No abstract

The structure of a new antifreeze protein (AFP) variant, RD3, from antarctic eel pout (Rhigophila dearborni) with enhanced activity has been determined for the first time by nuclear magnetic resonance spectroscopy. RD3 comprises a unique translational topology of two homologous type III AFP globular domains, each containing one flat, ice binding plane. The ice binding plane of the N domain is located ϳ3.5 Å "behind" that of the C domain. The two ice binding planes are located laterally with an angle of 32 ؎ 12°between the planes. These results suggest that the C domain plane of RD3 binds first to the ice {101 0} prism plane in the ͗0001͘ direction, which induces successive ice binding of the N domain in the ͗0101͘ direction. This manner of ice binding caused by the unique structural topology of RD3 is thought to be crucial for the significant enhancement of antifreeze activity, especially at low AFP concentrations.

No abstract

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required, A direct overexpression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of ~SN-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.