Defects of collagen XVII, a keratinocyte adhesion protein, are associated with epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant collagen XVII domains. The mutation occurred in a junctional epidermolysis bullosa patient who was compound heterozygous for the novel glycine substitution mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRNA was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine residues within the collagen triple helices are important for stable conformation, the thermal stability of the wild-type and mutated eukaryotic recombinant Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition, Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnormal triple-helix folding and susceptibility to proteolysis. Consistently, immunoassays demonstrated reduced amounts of the full-length collagen XVII and absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observations show that the glycine substitution G633D in collagen XVII causes abnormal folding and susceptibility to degradation, and thus perturbs the physiologic adhesive functions of collagen XVII in the skin.