Intrastrand Cross-Linked Actin between Gln-41 and Cys-374. I. Mapping of Sites Cross-Linked in F-actin by N-(4-azido-2-nitrophenyl) Putrescine

• Published in: Biochemistry (Easton) Vol. 37; no. 51; pp. 17784 - 17792
• Main Authors: Hegyi, György, Mák, Marianna, Kim, Eldar, Elzinga, Marshall, Muhlrad, Andras, Reisler, Emil
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.12.1998
• Subjects: Actins - chemistry; Actins - metabolism; Animals; Azides - metabolism; Calcium - metabolism; Computer Simulation; Cross-Linking Reagents - metabolism; Cysteine - metabolism; Glutamine - metabolism; Magnesium - metabolism; Mass Spectrometry; Models, Molecular; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Peptide Mapping; Photoaffinity Labels - metabolism; Putrescine - analogs & derivatives; Putrescine - metabolism; Rabbits; Sequence Analysis; Titrimetry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981285j

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40−50 (or 40−62 in the arginine limited digest) and residues 373−375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 Å, constrains to that range the distance between the γ-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44−49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826−836]. The effect of cross-linking on the function of actin is described in the companion papers.