Marfan syndrome is a heritable disorder of connective tissue which has been associated with mutations in a gene encoding fibrillin, a 350 kD glycoprotein found in microfibrils. This protein consists of approximately 60 domains, 47 of which have similarity to epidermal growth factor (EGF). The first mutations to be detected were found in two sporadic cases that had identical Arg to Pro changes within one EGF-like domain. Based on sequence features common to 43 of the EGF-like domains, it was proposed that these domains might bind calcium. Through the synthesis and characterization of wild-type and mutated single domain peptides, we examined the structural and calcium-binding properties of an isolated EGF-like domain from fibrillin and the effects of the Arg to Pro sequence change. A peptide corresponding to the thirteenth putative calcium-binding, EGF-like domain of fibrillin (the site of the first detected mutations) was synthesized. This peptide could be easily oxidized and refolded. The structure of this domain was probed using NMR methods, indicating features characteristic of the known structures of EGF-like domains. The domain bound to calcium with moderate affinity (Kd = 0.6 +/- 0.1 mM) with no major changes in structure induced upon calcium binding. A synthetic peptide containing the Arg to Pro mutation was found to be drastically impaired in its ability to fold in vitro. As predicted, a fibrillin domain forms a calcium-binding, EGF-like module. As the putative calcium-binding sites are found at the amino-terminal end of the modules, we propose that calcium ions may bind in the interfaces between domains, affecting the overall structure of the protein. The Arg to Pro mutation blocks domain folding in vitro, suggesting that lack of proper domain folding in vivo may contribute to the molecular defects responsible for Marfan syndrome.