peptide models consistent with nmr derived distance restraints were obtained by sequential application of restrained molecular dynamics and minimization using the powell method powell m j d 1977 conformational searches were carried out using a simulated annealing protocol the apomsh structures were heated to 2000k then cooled to 300k metal bound structures were only heated to 800k due to the highly restrained nature of the metal center structures emerging from simulated annealing runs were minimized to convergence gradient lt 0 05 kcal mol u00e5 and compared using the fit algorithm within sybyl peptide bioactivity was determined by measuring melanin released into cell culture media in an in vitro assay using b16f1 murine melanoma cells siegrist w 1986 cells were incubated with various concentrations of peptide and assayed for melanin production by measuring the absorbance of the culture media at 405 nm absorbance values were compared to a standard curve obtained using synthetic melanin from sigma the standard curve was linear over the experimental range of absorbance values quantitative receptor binding assays were carried out following a previously described method siegrist w 1988 sahm u g 1994 cells were seeded at a density of 5 u00d7 10 5 cells per well in 24 well tissue culture plates and exposed to varying concentrations of peptides on 0 5 ml of binding media 25 mm hepes naoh ph 7 4 0 2 bsa 0 3 mm 1 10 phenanthroline and approximately 100 000 cpm of 125 i nle 4 d phe 7 u03b1 msh 36 at 4 u00b0 c for 8 hours the cells were rinsed with pbs lysed with 1 m naoh and counted in a 1275 minigamma gamma counter from lkb wallac six.