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...
The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-AlaHyp triplets at aa 868 -876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T m of 2°C for helices with one mutant chain and a 6°C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with pro␣1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.
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