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Alanine‐scanning Mutagenesis of Human Interleulcin‐11: Identification of Regions Important for Biological Activity
Author(s) -
CZUPRYN MARTA,
BENNETT FRANN,
DUBE JENNIFER,
GRANT KATHY,
SCOBLE HUBERT,
SOOKDEO HEMCHAND,
McCOY JOHN M.
Publication year - 1995
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1995.tb32323.x
Subject(s) - alanine scanning , mutagenesis , alanine , directed mutagenesis , mutation , site directed mutagenesis , biology , protein folding , protein structure , microbiology and biotechnology , fusion protein , biochemistry , chemistry , amino acid , gene , recombinant dna , mutant
We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.