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 interleukin-6 lacking the 22 N-terminal residues (DeltaN22mIL-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 DeltaN22mIL-6, F173A lacks biological activity. F173A retains high levels of secondary structure, as determined by far-UV circular dichroism (CD), but has substantially reduced levels of tertiary structure, as determined by near-UV CD and (1)H NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) over a range of pH values and exhibits noncooperative equilibrium unfolding (as judged by the noncoincidence of monophasic unfolding transitions monitored by far-UV CD and lambda(max), with midpoints 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 properties of molten globule states, suggesting that the loss of activity of F173A results from the disruption of the fine structure of the protein, rather than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformation is no more susceptible to proteolytic attack than the parent protein. By analogy with human IL-6, Phe173 in DeltaN22mIL-6 makes multiple interhelical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.
Matthews
J M
JM
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Tumour Biology Branch), P.O. Box 2008, Royal Melbourne Hospital, Parkville 3050, Victoria, Australia.
Norton
R S
RS
Hammacher
A
A
Simpson
R J
RJ
D000409:Alanine / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D015850:Interleukin-6; D011994:Recombinant Proteins; D010649:Phenylalanine; D014508:Urea; D000409:Alanine