The structure of [(Boc-Cys(1)-Pro-Leu-Cys(4)-OMe)(S-tert-C4H9)Hg](-) (Boc: butoxycarbonyl), 1, was studied in N,V-dimethylformamide (DMF) and compared with that of [(Boc-Cys(1)-Pro-Leu-Cys(4)-OMe)Hg], 2, in order to discuss the intrinsic structural feature of the cysteine-containing metal-binding sites of proteins: Cys(i)-X-Y-Cys(i+3)/M2+ 1 was generated by the reaction of 2 with NaS-tert-C4H9. The geometry of the mercury ion (Hg2+) in 1 was proposed to be trigonal planar by UV-vis spectroscopy and Hg L(III) edge X-ray absorption fine structure (XAFS) measurements. Extended X-ray absorption fine structure (EXAFS) calculations yielded r(Hg-S) = 2.42 Angstrom. Analyses of the nuclear Overhauser and exchange spectroscopy (NOESY) and the rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of 1 in DMF-d(7) gave approximate distances for the 21 H-1-H-1 pairs of the main chain loop. These results on distance information were processed by distance geometry (DG) and restrained molecular dynamics (RMD) calculations in order to optimize the molecular structure of 1. Molecular dynamics (MD) calculations were also performed. We proposed that the trigonal planar Hg2+ in 1 regulates the hydrogen-bonding schemes of the peptide in the same manner as the tetrahedral ions involved in the Cys(i)-X-Y-Cys(i+3)/M2+ core sites in natural proteins, forming two hydrogen bonds, Cys(1) S-Leu H-N and Cys(1) S-Cys(4) H-N. This is in contrast to 2, where the linear coordinate mercury causes another type of hydrogen-bonding scheme, Cys(1) S-Leu H-N and Pro CO-Cys(4) H-N. Details of the effect of trigonal planar Hg2+ on the peptide conformation were analyzed with respect to the phi, phi, and chi torsion angles of the peptide chain. The effect of the change of the angle S-Hg-S bite angle on the conformation of Cys-Pro-Leu-Cys was also discussed on the basis of MD calculations. The distribution area of Leu (phi, psi) in the Ramachandran plot moves from near the ct helix region to the turn structure region as the bite angle increases from 90 to 180 degrees, accompanying the change in the hydrogen-bonding scheme. The critical bite angle is around 140 degrees. The analysis revealed that angle S-Hg-S congruent to 110 degrees, which corresponds to the tetrahedral coordination geometry of the central metal ion, allows a high flexibility of the Cys-Pro-Leu-Cys skeleton.