Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine

Abstract

The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the y-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-I13 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nuture 347, 44-49), the a-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.

Beryllium Fluoride and Phalloidin Restore Polymerizability of a Mutant Yeast Actin (V266G,L267G) with Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop

“…This model agrees well with the structural model derived from other data fitting techniques (6,7) and optical reconstruction of electron micrographs of actin filaments (8 -10). The orientation of the monomer in the helix in this model agrees with the results of a genetic study of the F-actin binding site of phalloidin (11), chemical cross-linking experiments (12), and antibody decoration experiments (13,14). However, there is little evidence concerning the validity of the model pertaining to the intermonomer contacts that stabilize the helix.…”

Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274

“…The hydrophobic plug at the tip of the loop can now insert into a hydrophobic pocket formed by the interface of two monomers on the opposing strand of the helix, leading to an appreciable increase in the cross-strand stabilization available for the actin filament. Good agreement exists between this model and the results of low resolution structural studies (8 -10), protein chemical studies (11), and antibody decoration studies (12)(13). However, the model remains in large part unproven.…”

Identification of Cytoplasmic Actin as an Abundant Glutaminyl Substrate for Tissue Transglutaminase in HL-60 and U937 Cells Undergoing Apoptosis

“…The lack of DALP labeling of other abundant proteins of the cellular machinery (such as tubulin) also points to the specificity of actin labeling. It was also demonstrated that Gln-41 is the main amine acceptor site for the transglutaminase reaction (39,40), and the enzyme can also form trace amounts of covalently cross-linked actin oligomers from G actin, which may act as "seeds" to allow actin to polymerize faster. Furthermore, since Gln-41 is accessible in F actin as well (39) and according to our present results the high molecular weight polymers formed by TGase are also labeled by DALP, it is very likely that additional proteins are attached covalently, through their accessible Lys residues, to the microfibrillar meshwork by the enzyme.…”