Abstract
The relationship between protein sequences and their conformation was examined by the effects of sequence orientation upon the structure. Both theoretical and experimental approaches were used to explore the significance of the direction of peptide backbone. In comparison with its parent peptide sequence, the reverse peptide sequence achieves a mirroring side chain position, a exchange of C=O and N-H groups of backbone, and an the oriental change of backbone dipole moment. Among the proteins examined, which include B domain of Staphylococcal protein A, B1 domain of Staphylococcal protein G, SH3 domain of a-spectrin, zinc finger, and a domain of human metallothionein-2 (MT-2), theoretical calculations showed that the backbone dipole moment of a domain of metallothionein is the minimum. Since MT-2 a domain is known to be able to form a metal cluster with the cysteine side chains as a folding nucleus. It is implied that the retro a domain of metallothionein might be foldable. The backward reading sequence of human metallothionein-2 a-domain was synthesized and its chemical and spectroscopic properties analyzed. This retro-a-domain was shown to bind Cd(II) in identical stoichiometries with the chemically synthesized a-domain of metallothionein-2. Nearly identical to the a-domain, Cd-binding retro-a-domain showed a characteristic ultraviolet absorption spectrum with a shoulder at 245-250 nm due to cadmium-thiolate charge transfer, and the absorption shoulder was abolished by acidification, suggesting a mercaptide bonding between Cd(II) and the cysteine residues. Similar metal-binding capabilities between a- and retro-a-domains were observed also by pH titration and in the reaction with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid). A two-state cooperativity of the metal-cluster formation was observed spectroscopically in the titration of the retro-a-domain, indicating that the retro-protein is a foldable protein. These results suggest that the order of the amino acid sequence for a metal binding domain in a given metalloprotein is not critical to the foldibility and metal-binding capacity, contrasting to those of other proteins, providing that critical residues for metal binding are present. However, circular dichroism spectra of the Cd-binding a- and retro-a- domains showed that the reversal direction of the domain sequence backbone significantly affects the formation of structure even it is foldable.