Sequence 18-29 on actin: antibody and spectroscopic probing of conformational changes.
Experimental evidence for the involvement of the 18-29 site within actin subdomain-1 in the actomyosin weak binding interface includes the inhibition of actomyosin ATPase activity by specific peptide antibodies [Adams, S., & Reisler, E. (1993) Biochemistry 32, 5051-5056] and by the Dictyostelium actin mutant D24H/D25H [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In this work, the effect of the 18-29 peptide antibodies on the polymerization and conformation of actin has been characterized. Binding of antibody to the 18-29 site strongly inhibited the MgCl2-induced polymerization of G-actin, had a much weaker impact on the CaCl2 polymerization of actin, and showed very little effect on the NaCl polymerization of G-actin. These observations were linked to the binding of the 18-29 antibody to the different forms of actin. In sedimentation assays, the (18-29) IgG bound more strongly to Mg-F- and Mg-G-actins than to Ca-F- and Ca-G-actins, respectively. The binding of IgG to F-actin decreased sharply with an increase in ionic strength. Antibody binding to the 18-29 site induced conformational changes within the nucleotide cleft, both slowing the rate of nucleotide exchange and increasing the fluorescence intensity of actin-bound epsilon ATP. The increased fluorescence of a dansyl probe attached to Gln-41 and a pyrene probe attached to Cys-374 demonstrated that antibody binding also caused local perturbations in the DNase I loop of subdomain-2 and at the C-terminus of actin. These results are discussed in terms of actin plasticity and its implications for actomyosin interactions.
Adams
S B
SB
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007158:Immunologic Techniques; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011108:Polymers; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D012965:Sodium Chloride / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D007074:Immunoglobulin G; D011108:Polymers; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium