Matrix proteins from insect pliable cuticles: are they flexible and easilydeformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk m oth larvae, Hyalophora cecropia, were studied in solution by means of a flu orescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in nonpolar media than in polar media. An intense ANS-fluo rescence was observed in the presence of the cuticular proteins at pH-value s close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denat urants were added. Aggregation and eventual precipitation of both H. cecrop ia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A d ecreased ANS-fluorescence was observed at salt concentrations too low to ca use visible aggregation of the H. cecropia proteins, probably due to weaken ed electrostatic interactions between chain segments, but such a decrease w as not observed for the locust proteins. The changes in intensity of ANS-fl uorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are expos ed to the solvent, when either hydrophobic interactions or electrostatic at tractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are availa ble for protecting ANS-molecules from the quenching effects of water. The e ffects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as h en egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed h ydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation correspondi ng to that of globular proteins. The behaviour of the cuticular proteins re sembles that decribed for unordered, randomly coiled, thermally agitated po lymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cu ticular proteins are in an unordered, random structure both when the protei ns are in solution and when present in the intact cuticle; probably only th e chain regions involved in binding the proteins to chitin will have a well -defined spatial organisation. (C) 2001 Elsevier Science Ltd. All rights re served.