Deletion of amino acids from the carboxy‐terminal end of actin

Abstract

A series of deletions was made from the C-terminal end of actin by inserting termination codons into a full length cDNA of human alpha-skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNAs were translated in vitro using 35S-labeled methionine. The 35S-labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S-1, bind a 27 kDa proteolytic fragment of alpha-actinin, and incorporate into myofibrils in vitro. Removal of the C-terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S-1 or the 27 kDa fragment of alpha-actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356-365 is due to the important role Trp356 plays in maintaining hydrophobic bonds between three non-contiguous segments of actin. We also suggest that removal of residues 366-372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibrils.

Plasmodium actin is incompletely folded by heterologous protein‐folding machinery and likely requires the native Plasmodium chaperonin complex to enter a mature functional state

“…In each study, C-terminal truncations have also been shown to increase the critical concentration, polymerization rate, and rate of ATP hydrolysis of the filaments (47-49). C-terminal truncations have also been shown to lead to conformational changes in the D-loop of SD2, disrupting the interactions between neighboring monomers in the filament, affecting filament stability, and causing observed structural changes to the actin filament (49)(50)(51)(52)(53). Finally, in vivo, truncation of the last 2 residues, C374 and F375, of yeast actin (the last 3 are lethal) leads to a complete loss of actin filamentous structures (54) with a similar effect seen in mammalian cells containing actin having the same mutated C374, which led to a disruption and disorganization of observable filamentous structures with an accompanied increase in diffuse actin staining (55).…”

The Cytosolic Class II Chaperonin CCT Recognizes Delineated Hydrophobic Sequences in Its Target Proteins

“…This indicates that the N-terminal parts of these two CCTtarget proteins are necessary for correct folding (or for stability) despite their being dispensable for binding to CCT. Xia and Peng (42) reported that region 356-365 plays a key role in maintaining the structural integrity of actin. This is in agreement with our observation that removal of the 25 C-terminal residues results in an increased CCT binding (compared to full-length actin) due to the incapability of this fragment of reaching a correctly folded state.…”

Actin Interacts with CCT via Discrete Binding Sites: A Binding transition-release Model for CCT-Mediated Actin Folding

“…19,39 Therefore, one of the CCT functions may be to assist actin in bringing its C terminus close to its N terminus. On the basis of EM studies, actin transits from an open to a closed state.…”