The folding and assembly pathway of tumour necrosis factor TNF alpha, a globular trimeric protein

The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-alpha (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric intermediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-1-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.