Aggregation of wool keratin intermediate filament proteins.
The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.
Woods
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EF
CSIRO, Division of Wool Technology, Parkville, Australia.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000737:chemistry*; D007633:Keratins / Q000032:analysis / Q000737:chemistry*; D008970:Molecular Weight; D011487:Protein Conformation; D012756:Sheep; D014461:Ultracentrifugation; D014935:Wool / Q000737:chemistry*
D000596:Amino Acids; D007381:Intermediate Filament Proteins; D007633:Keratins