Induction of α-Helix in the β-Sheet Protein Tumor Necrosis Factor-α: Acid-Induced Denaturation

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Abstract
Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or “A” state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for α-helical proteins, while few studies have been done on proteins containing only β-strands. Tumor necrosis factor-α (TNF-α) is a trimer in which the individual subunits consist of antiparallel β-sheet, organized into a jellyroll β-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447−11453] that thermal denaturation of TNF-α results in an aggregate which contains a substantial amount of α-helix and that the addition of trifluoroethanol induces α-helix in both murine and human TNF-α. Here we show that acid also can induce α-helix in these proteins. At acidic pH (below 4), both human and murine TNF-α convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-α is a molten globule. What is unique in this protein is that TNF-α acquires α-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-α at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in α-helical content. At pH 2.2 in the absence of salt, TNF-α shows considerable α-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of α-helix at ambient temperature, and upon heating, we see the noncooperative increase in α-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an α-helical monomer of TNF-α may be related to its known interaction with lipid bilayers.