INDUCTION OF ALPHA-HELIX IN THE BETA-SHEET PROTEIN TUMOR-NECROSIS-FACTOR-ALPHA - THERMAL-INDUCED AND TRIFLUOROETHANOL-INDUCED DENATURATION AT NEUTRAL PH

The unfolding and refolding of alpha-helical proteins has been extensi vely studied, demonstrating formation of intermediate structures which retain the native-like alpha-helix but lack the tertiary structure. S tudies on the folding of proteins consisting primarily of beta-sheet a re interesting since, unlike the alpha-helix, the beta-sheet requires the formation of peptide hydrogen bonds between two or more polypeptid e segments which may be far apart in the linear sequence. Here we have studied the unfolding of the beta-sheet-containing protein tumor necr osis factor-alpha (TNF-alpha). This protein exists as a symmetric trim er in solution. Murine TNF-alpha begins to melt at 60 degrees C and un folds to a soluble structure with a transition midpoint of 66 degrees C. This reaction is irreversible. This unfolded form contains a consid erable amount of (similar to 30%) alpha-helix, as determined by circul ar dichroism. Human TNF-alpha begins to melt at 60 degrees C and preci pitates concurrently with unfolding, such that there is no soluble pro tein present by 70 degrees C. The secondary and tertiary structures of murine TNF-alpha unfold simultaneously, suggesting that unfolding fro m the native to the unfolded state occurs cooperatively. The thermal-i nduced denaturation is very insensitive to protein concentration, indi cating that trimer to monomer conversion, if it occurs, is not rate-li miting. Trifluoroethanol induces alpha-helix in both human and murine TNF-alpha, further demonstrating the propensity of TNF-alpha to form a lpha-helix. The different behavior of human versus murine TNF-alpha up on thermal unfolding is due to differences in the solubility of the un folded protein, the murine form being more soluble. These results indi cate that TNF-alpha can form alpha-helix when the long range interacti ons conferred by the native structure are removed during unfolding.