Measured by ultra-slow scanning calorimetry and isothermal circular dichroism, human lung collagen monomers denature at 37°C within a couple of days. Their unfolding rate decreases exponentially at lower temperature, but complete unfolding is observed even below 36°C. Refolding of full-length, native collagen triple helices does occur, but only below 30°C. Thus, contrary to the widely held belief, the energetically preferred conformation of the main protein of bone and skin in physiological solution is a random coil rather than a triple helix. These observations suggest that once secreted from cells collagen helices would begin to unfold. We argue that initial microunfolding of their least stable domains would trigger self-assembly of fibers where the helices are protected from complete unfolding. Our data support an earlier hypothesis that in fibers collagen helices may melt and refold locally when needed, giving fibers their strength and elasticity. Apparently, Nature adjusts collagen hydroxyproline content to ensure that the melting temperature of triple helical monomers is several degrees below rather than above body temperature.T ype I collagen is the most abundant animal protein, and forms the matrix of bone, skin, and other tissues. One would think that matrix proteins should be very stable. Nevertheless, for over half a century it was taken for granted that triple helices of type I collagen melt just several degrees above body temperature (1, 2). Naturally, much attention was paid to the origins (3, 4) and physiological role (5) of this marginal thermal stability. It was proposed, for example, that destabilization of type I collagen by mutations is an important factor in osteogenesis imperfecta, a debilitating and often lethal hereditary disorder characterized by brittle bones (6, 7).Most measurements of collagen denaturation were (and still are) done by scanning at Ϸ0.02-2°C͞min heating rate or after a short equilibration at constant temperature (usually several minutes). To infer the equilibrium melting temperature, T m , one must extrapolate such data to zero heating rate or infinite waiting time (1). But a recent differential scanning calorimetry (DSC) study (8) and our DSC measurements at much slower rates ( Fig. 1) unequivocally show that the apparent T m changes linearly with the logarithm of the heating rate at all rates and equilibration times reported before. Because a logarithmic dependence cannot be extrapolated to zero argument (Fig. 1), the equilibrium T m cannot be inferred from the published data. Either collagen denaturation is an intrinsically nonequilibrium process (8) or the equilibrium T m is lower and the protein is less stable than previously believed.In the present study we demonstrate that the equilibrium T m of collagen does exist, but it is several degrees below body temperature in physiological solution. The thermodynamically preferred conformation of collagen at body temperature is a random coil rather than helix. This must be a deliberate design, because Nature tunes collag...

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.

Classic osteogenesis imperfecta, an autosomal dominant disorder associated with osteoporosis and bone fragility, is caused by mutations in the genes for type I collagen. A recessive form of the disorder has long been suspected. Since the loss of cartilage-associated protein (CRTAP), which is required for post-translational prolyl 3-hydroxylation of collagen, causes severe osteoporosis in mice, we investigated whether CRTAP deficiency is associated with recessive osteogenesis imperfecta. Three of 10 children with lethal or severe osteogenesis imperfecta, who did not have a primary collagen defect yet had excess post-translational modification of collagen, were found to have a recessive condition resulting in CRTAP deficiency, suggesting that prolyl 3-hydroxylation of type I collagen is important for bone formation.