The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Abstract

The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-a (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric interrnediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-I-naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid-induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.

Activity of Tumor Necrosis Factor α Is Modulated by Dynamic Conformational Rearrangements

“…However, the strong interaction of SPD304 and ANS precluded a quantitative comparison of the two perturbations of the PPI. For Y119S, the effect remains much lower than for TNFα under conditions reported to mimic a molten globular TNFα (1 M guanidinium chloride) . Interestingly, Y59A does not induce such a shift, in agreement with previous NMR experiments indicating that this mutation induces less severe perturbation of the PPI than Y119S.…”

“…The idea that TNFα can exist as a trimer with increased flexibility and dynamics was previously suggested in studies on the folding pathway of TNFα, where the disassembly of TNFα happens via an intermediate trimeric molten globule. , Our analysis reveals that TNFα has some but limited characteristics of a molten globule-like state, resulting from loosening of the PPI while no evidence of significant loss of tertiary or quaternary structure could be observed.…”

Probing the solution structure of tumor necrosis factor‐α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry

“…The TNF‐ α was at a concentration of 1 mg/ml, which is in the micromolar range. This concentration has been shown to promote trimer formation (Corti et al , ; Alzani et al , ; Hlodan and Pain, ). The TNF‐ α was solubilized in 3.5 m m NH 4 OAc, pH 5.5 buffer with no acids or organics for all non‐covalent interaction analyses.…”

“…Therefore, shifting the pH to either lower than 5.0 or higher than 9.0 to shift the equilibrium toward monomer formation was the first choice for these studies. However, it was unclear what pH would only partially, verses completely, dissociate the trimer complex (Hlodan and Pain, ). The ability to differentially dissociate a protein complex would be useful for generating various heterotrimers, as well as to have control over the degree of complex dissociation.…”

“…There are a number of reports in the literature that examine the trimer association as well as its stability. The dissociation/re‐association of the TNF‐ α trimer as a function of concentration has been documented extensively (Kunitani et al , ; Corti et al , ; Alzani et al , ; Hlodan and Pain, ; Ameloot et al , ). In addition, monomer/trimer ratios have been found to fluctuate with temperature (Kunitani et al , ).…”