Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.
We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities.
Orlova
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Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis 55455.
Yu
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Egelman
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D000199:Actins / Q000737:chemistry* / Q000276:immunology* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000648:ultrastructure; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004923:Erythrosine; D007091:Image Processing, Computer-Assisted; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D008854:Microscopy, Electron; D008958:Models, Molecular; D015879:Myosin Subfragments / Q000737:chemistry; D010590:Phalloidine; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D015879:Myosin Subfragments; D010590:Phalloidine; D000255:Adenosine Triphosphate; D004923:Erythrosine