TY - JOUR
T1 - 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
AU - Hegyi, György
AU - Mák, Marianna
AU - Kim, Eldar
AU - Elzinga, Marshall
AU - Muhlrad, Andras
AU - Reisler, Emil
PY - 1998/12/22
Y1 - 1998/12/22
N2 - 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 373375, 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.
AB - 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 373375, 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.
UR - http://www.scopus.com/inward/record.url?scp=0032558990&partnerID=8YFLogxK
U2 - 10.1021/bi981285j
DO - 10.1021/bi981285j
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C2 - 9922144
AN - SCOPUS:0032558990
SN - 0006-2960
VL - 37
SP - 17784
EP - 17792
JO - Biochemistry
JF - Biochemistry
IS - 51
ER -