The single mutation Phe173 -> Ala induces a molten globule-like state in murine interleukin-6

A series of three aromatic to alanine mutants of recombinant murine interle ukin-6 lacking the 22 N-terminal residues (Delta N22mIL-6) were constructed to investigate the role of these residues in the structure and function of mIL-6. While Y78A and Y97A have activities similar to that of Delta N22mIL -6, F173A lacks biological activity. F173A retains high levels of secondary structure, as det-ermined by far-UV circular dichroism (CD), but has subst antially reduced levels of tertiary structure, as determined by near-UV CD and H-1 NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8- naphthalenesulfonic acid (ANS) over a range of pH values and exhibits nonco operative equilibrium unfolding (as judged by the noncoincidence of monopha sic unfolding transitions monitored by far-UV CD and lambda(max), with midp oints of unfolding at 2.6 +/- 0.1 and 3.5 +/- 0.3 M urea, respectively, and the lack of an observable thermal unfolding transition). These are all pro perties of molten globule states, suggesting that the loss of activity of F 173A results from the disruption of the fine structure of the protein, rath er than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformat ion is no more susceptible to proteolytic attack than the parent. protein. By analogy with human IL-6, Phe173 in Delta N22mIL-6 makes multiple interhe lical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.