Type I collagen is thermally unstable at body temperature - PubMed (original) (raw)

Type I collagen is thermally unstable at body temperature

E Leikina et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Measured by ultra-slow scanning calorimetry and isothermal circular dichroism, human lung collagen monomers denature at 37 degrees C within a couple of days. Their unfolding rate decreases exponentially at lower temperature, but complete unfolding is observed even below 36 degrees C. Refolding of full-length, native collagen triple helices does occur, but only below 30 degrees 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.

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Figures

Figure 1

Figure 1

Thermal denaturation of rat-tail-tendon collagen measured by DSC in 0.2 M phosphate/0.5 M glycerol (pH 7.4), unbuffered 2 mM HCl (pH 2.7), and 50 mM acetic acid (pH 2.7). The temperatures in the phosphate/glycerol buffer are corrected to represent physiological conditions by subtraction of 1.7°C (see Materials and Methods). (a) Raw DSC data in phosphate/glycerol buffer at different heating rates. To simplify visual comparison, the base lines were subtracted and the data were normalized to ensure the same height of the denaturation peak. (b) Dependence of the temperature (_T_m) at the maximum of the denaturation peak on the heating rate and (c) on the logarithm of the heating rate. Because the apparent _T_m is a linear function of the logarithm of the heating rate rather than of the rate itself, calculation of the equilibrium _T_m by extrapolation of this dependence to zero heating rate is impossible (formally it would give _T_m = −∞).

Figure 2

Figure 2

Thermal denaturation of rat-tail-tendon and human-lung collagens in 0.2 M sodium phosphate/0.5 M glycerol, pH 7.4 (all temperatures corrected by −1.7°C to represent physiological conditions). (a) Isothermal measurement of denaturation kinetics from CD spectra. Fraction of native protein is calculated from the ellipticity at 221 nm by comparing it with the ellipticities of the solution before the experiment and after complete denaturation of the protein. (b) Fraction of native protein vs. equilibration time in isothermal measurements. (c) DSC (circles) and isothermal CD (squares) measurement of apparent melting temperature (_T_m) vs. equilibration time. In isothermal CD, _T_m is the temperature of the experiment and the equilibration time is the time at 50% denaturation. In DSC, _T_m is the temperature at the maximum of the melting peak and the equilibration time is recalculated from the heating rate (see Materials and Methods).

Figure 3

Figure 3

Renaturation of type I rat-tail-tendon collagen in 0.2 M sodium phosphate/0.5 M glycerol at pH 7.4 (all temperatures corrected by −1.7°C as in Fig. 2 for consistency with denaturation data). (a) Recovery of the triple helical CD signal on renaturation at different temperatures. (b) Dependence of the renaturation half-time (the time needed to recover 30% helicity) on the temperature. (c) DSC of native collagen solution (black) and solutions renatured at different temperatures (blue, green, and red). DSC of fibrillogenesis-incompetent (cyan) and fibrillogenesis-competent (magenta) fractions of collagen renatured at 24°C and digested by pepsin for 1 h at 35°C in 0.5 M acetic acid. (d) SDS gel electrophoresis of the same fibrillogenesis-competent (lane 1) and -incompetent (lane 2) fractions and of native (lane 3) collagen. Because only full-length helices could resist pepsin cleavage at 35°C, the fibrillogenesis-competent fraction consists mostly of α13 and a much smaller amount of α12α2 helices [intact α2(I) chains do become visible in this fraction on gel overloading]. Judging from the relative intensities of α1 and α2 bands, fibrillogenesis-incompetent fraction consists mostly of α23 and some α1α22 helices.

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