Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?
Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that described for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.
August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 O, Copenhagen, Denmark. soandersen@aki.ku.dk
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D000817:Anilino Naphthalenesulfonates; D000818:Animals; D005456:Fluorescent Dyes; D006110:Grasshoppers / Q000502:physiology*; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins; D009036:Moths / Q000502:physiology*; D018583:Pliability; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D019476:Insect Proteins; C027132:1-anilino-8-naphthalenesulfonate; D019791:Guanidine