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Biochemistry. 2004 Jun 29;43(25):8021-8.

Synthetic cross-links arrest the C-terminal region of the relaxin-like factor in an active conformation.

Author information

1
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250509, Charleston, South Carolina 29425, USA. bullesee@musc.edu

Abstract

All kinetic indicators suggest that the receptor-binding site of the relaxin-like factor (RLF) is located in the flexible C-terminal region of the B chain and is centered about the tryptophan in position B27. Conformational restraints of varying lengths were used to confine Trp (B27) to a narrow range of positions relative to the C terminus of the A chain. Total synthesis of variants involving residues proximate to Trp (B27) assured us that none had a role in receptor binding. Even arginine B26, next to the important tryptophan, can be replaced without deleterious effects. To fix the distance between the C-terminal end of the A chain and Trp (B27) at predetermined lengths, we synthesized RLF with covalent cross-links between a lysine, which was placed in position B26, and the alpha-carboxyl group at the C terminus of the A chain (A26). The affinity of the cross-linked ligands for the RLF receptor varied as a parabolic function of length whereby the range of 10.0-11.1 A provided the closest approach to the binding conformation. Wild-type transmembrane signaling activity, as determined by cAMP accumulation, was reached only with a glycine cross-linker.

PMID:
15209497
DOI:
10.1021/bi049601j
[Indexed for MEDLINE]

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