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

Biochemistry. 2004 Jun 29;43(25):8021-8. doi: 10.1021/bi049601j.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Circular Dichroism
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / pharmacology*
  • Cyclic AMP / metabolism
  • Dose-Response Relationship, Drug
  • Female
  • Humans
  • Insulin
  • Lysine / chemistry
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Conformation
  • Proteins / chemical synthesis
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Receptors, G-Protein-Coupled
  • Receptors, Peptide / genetics
  • Receptors, Peptide / metabolism
  • Spectrometry, Fluorescence
  • Transfection
  • Tryptophan / chemistry
  • Uterus / metabolism


  • Cross-Linking Reagents
  • Insulin
  • Leydig insulin-like protein
  • Proteins
  • Receptors, G-Protein-Coupled
  • Receptors, Peptide
  • relaxin receptors
  • Tryptophan
  • Cyclic AMP
  • Lysine