Analysis of the phasing of four spectrin-like repeats in alpha-actinin.
Selected fragments of the central rod of chicken gizzard alpha-actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin-like repeats that make up this domain. The criteria for an independently folding unit were resistance to proteolysis and the high alpha helicity characteristic of the native protein. Sequences containing repeats 1-4, 2-4, 3-4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N-terminal sequencing gave the most probable starting position of each repeat. The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease-resistant fold in solution. The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N-terminal boundaries of the repeats were 14-24 residues nearer the C-terminus than predicted. The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of beta 1 integrins and intercellular cell adhesion molecule-1 which have been localised to the rod domain of alpha-actinin.
Gilmore
A P
AP
Department of Biochemistry, University of Leicester, England.
Parr
T
T
Patel
B
B
Gratzer
W B
WB
Critchley
D R
DR
D000185:Actinin / Q000096:biosynthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli; D005895:Gizzard, Avian; D005982:Glutathione Transferase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry*; D013820:Thermolysin; D014357:Trypsin
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000185:Actinin; D013049:Spectrin; D005982:Glutathione Transferase; D014357:Trypsin; D013820:Thermolysin