Structural studies of antifungal protein, tenecin 3 by CD and NMR

Abstract

Structural characteristics of antifungal protein, tenecin 3 has been investigated by CD and NMR in various condition. Tenecin 3 is composed of 78 amino acids and rich in glycine, glutamine and histidine. These residues occupy about 80% of entire sequence. Interestingly, tenecin 3 has 11 times repeated motif of Gly-X-X-Gly or X-Gly-Gly-X, where X is glutamine, histidine or leucine. CD experiments of His tag fused tenecin 3 showed that its secondary structure is mainly composed of unordered form. Although high helicity was induced in high concentration of TFE, it seems to originate from His tag fragment. pH did not affect structural change. To correlate the activity of tenecin 3 with cytoplasmic membrane, SDS micelle and liposomes composed of PC or PC:PG(3:1) were constructed. It was found that no special structural change was observed in these environments, indicating that tenecin 3 may not act on cytoplasmic membrane. Enterokinse was treated on tenecin 3 to eliminate His tag fragment, but surprisingly it was found that cleaved His tag fused tenecin 3 show fair affinity to metal resin. This suggests that intact tenecin 3 can be highly purified by the same method. Fermentation of E. coli encoding intact tenecin 3 was carried out to obtain large amounts of protein. CD experiments of tenecin 3 gave similar results like previous CD experiments. The portion of random coil and turn were estimated by about 50% and 40% in most conditions, respectively. NMR experiments showed that random property is dominated in tenecin 3, resulting from the facts that any NH/NH cross-peak was not seen in NOESY and amide cross-peaks was highly overlapped. Chitin binding assay was performed to correlate its activity with chitin. Although tenecin 3 showed some binding affinity to chitin, it is not clear if it actually act on chitin. It was also found that tenecin 3 does not have chitinase activity.