C-13 NMR OF NEPHILA-CLAVIPES MAJOR AMPULLATE SILK GLAND

The major ampullate glands of the spider Nephila clavipes contain simi lar to 0.2 mu l each of a highly concentrated (similar to 50%) solutio n of silk fibroin. Therefore, the reservoir of silk in these glands pr esents an ideal opportunity to observe prefolded conformations of a pr otein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by C-13 NMR spectroscopy. These results wer e compared to those from silk protein first drawn from the spinneret a nd then denatured. The C-13 NMR chemical shifts, along with infrared a nd circular dichroism data, suggest that the silk fibroin in the gland s exists in dynamically averaged helical conformations. Furthermore, t here is no evidence of proline residues in U-C-13-o-glucose-labeled si lk. This transient prefolded ''molten fibril'' state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of th e final beta-sheet structure in the ampullate gland silk fibroin befor e final silk processing. However, the conformation of silk in the glan ds appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecti ng the ampullate glands to the spinnerets play a larger role in silk p rocessing than previously thought.