Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin.
Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000097:blood*; D002918:Chymotrypsin; D002942:Circular Dichroism; D018260:Gelsolin; D008840:Microfilament Proteins / Q000097:blood*; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D002918:Chymotrypsin; D002118:Calcium