Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.
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 gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-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.
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
G
G
Hegyi
H
H
Michel
J
J
Shabanowitz
D F
DF
Hunt
N
N
Chatterjie
G
G
Healy-Louie
M
M
Elzinga
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011700:Putrescine / Q000031:analogs & derivatives* / Q000737:chemistry; D011817:Rabbits; D011503:Transglutaminases / Q000378:metabolism
D000199:Actins; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D005973:Glutamine; C081612:N-(4-azidobenzoyl)putrescine; D011503:Transglutaminases; D008239:Lysine; D011700:Putrescine