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

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 alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha 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-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-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-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-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 alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.

Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

“…Recombinant human TNF purified from yeast or E. coli was shown to be present in its trimeric form in previous crystallographic 41 and AUC studies 42 . To confirm the oligomeric state of human TNF produced recombinantly in the baculovirus expression system used in this study, UV-SV AUC was performed using samples at a concentration of 2 μM.…”

Stability of the allergenic soybean Kunitz trypsin inhibitor

“…Acid commonly induces a molten state in proteins [19]. Subjecting SKTI to increasing acidity by addition of HCl clearly resulted in a diminished ellipticity in the far-UV CD spectrum.…”

“…Acidification can also result in the production of a molten globule structure [17], which is able to refold when the pH of the milieu is increased. a-Helical proteins have been studied extensively in terms of acid denaturation but relatively few reports exist on the acid denaturation of proteins containing only h-sheet structures [19].…”

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

“…Some organic solvents have been used earlier to change the conformation of protein molecules for studying protein folding/unfolding. For example, trifluoroethanol was used to induce formation of ␣-helices in various proteins such as ␤-lactoglobulin [7], porin [8] and tumor necrosis factor [9]. The alcohol-induced denaturation of some proteins e.g.…”