ABSTRACT

Copper complexes of β-casomorphin (BCM7 BCM5, BCM4) and other sequence related peptides (Tyr-Pro-Leu-Gly-NH₂, Tyr-Pro-Gly-Gly) were investigated by EPR and NMR in DMSO-d₆ solutions. Speciation of copper among many of the possible isomers was apparent. Computer simulations of low and room temperature EPR allowed the number of co-ordinated nitrogens in the major species to be inferred and a rotational correlation time of 0.18 ns at 298 K to be evaluated for all complexes. All isomers (but two of BCM7) were shown to bind copper, and the resulting structures were strictly determined by the conformational state of ²Pro. The trans rather than the cis conformation was shown to allow binding of the deprotonated ³X-NH; the terminal amino and carboxylate groups provided the other binding groups in all cases. Structures were obtained by constrained molecular dynamics using copper-proton distances obtained from paramagnetic nuclear relaxation rates. In the case of BCM7, only the cis-cis-trans and/or the cis-cis-cis isomers were not binding copper. The conformational state of each Pro was shown to drive formation of the copper-nitrogen bond within the immediately adjacent residue, leading to the complex having four co-ordinated nitrogens in the case of the trans-trans-trans isomer. Interpretation of paramagnetic relaxation rates of ²Pro-H_α signals provided the corresponding isomeric probabilities in the metal-bound state. The observed change in the values of existential probabilities upon copper addition may be relevant for the biological role of copper as well as of other metal ions.