BackgroundType I collagen is the most common protein among higher vertebrates. It forms the basis of fibrous connective tissues (tendon, chord, skin, bones) and ensures mechanical stability and strength of these tissues. It is known, however, that separate triple-helical collagen macromolecules are unstable at physiological temperatures. We want to understand the mechanism of collagen stability at the intermolecular level. To this end, we study the collagen fibril, an intermediate level in the collagen hierarchy between triple-helical macromolecule and tendon.Methodology/Principal FindingWhen heating a native fibril sample, its Young’s modulus decreases in temperature range 20–58°C due to partial denaturation of triple-helices, but it is approximately constant at 58–75°C, because of stabilization by inter-molecular interactions. The stabilization temperature range 58–75°C has two further important features: here the fibril absorbs water under heating and the internal friction displays a peak. We relate these experimental findings to restructuring of collagen triple-helices in fibril. A theoretical description of the experimental results is provided via a generalization of the standard Zimm-Bragg model for the helix-coil transition. It takes into account intermolecular interactions of collagen triple-helices in fibril and describes water adsorption via the Langmuir mechanism.Conclusion/SignificanceWe uncovered an inter-molecular mechanism that stabilizes the fibril made of unstable collagen macromolecules. This mechanism can be relevant for explaining stability of collagen.

We measured the Young's modulus at temperatures ranging from 20 to 100 degrees C for a collagen fibril that is taken from a rat's tendon. The hydration change under heating and the damping decrement were measured as well. At physiological temperatures 25 to 45 degrees C, the Young's modulus decreases, which can be interpreted as an instability of the collagen. For temperatures between 45 and 80 degrees C, the Young's modulus first stabilizes and then increases when the temperature is increased. The hydrated water content and the damping decrement have strong maximums in the interval 70 to 80 degrees C indicating complex intermolecular structural changes in the fibril. All these effects disappear after heat-denaturation of the sample at 120 degrees C. Our main achievement is a five-stage mechanism by which the instability of a single collagen at physiological temperatures is compensated by the interaction between collagen molecules.

Oxygen is released to living tissues via conformational changes of hemoglobin from R-state (oxyhemoglobin) to T-state (desoxyhemoglobin). The detailed mechanism of this process is not yet fully understood. We have carried out micromechanical experiments on oxyhemoglobin crystals to determine the behavior of the Young’s modulus and the internal friction for temperatures between 20 °C and 70 °C. We have found that around 49 °C oxyhemoglobin crystal samples undergo a sudden and strong increase of their Young’s modulus, accompanied by a sudden decrease of the internal friction. This sudden mechanical change (and the ensuing force release) takes place in a partially unfolded state and precedes the full denaturation transition at higher temperatures. After this transformation, the hemoglobin crystals have the same mechanical properties as their initial state at room temperatures. We conjecture that it can be relevant for explaining the oxygen-releasing function of native oxyhemoglobin when the temperature is increased, e.g. due to active sport. The effect is specific for the quaternary structure of hemoglobin, and is absent for myoglobin with only one peptide sequence.

Our micromechanical experiments show that at physiological temperatures type I collagen fibril has several basic features of the glassy state. The transition out of this state [softening transition] essentially depends on the speed of heating v, e.g., for v = 1 C/min it occurs around 70 C and is displayed by a peak of the internal friction and decreasing Young's modulus. The softening transition decreases by 45 C upon decreasing the heating speed to v = 0.1 C/min. For temperatures 20-30 C the native collagen fibril demonstrates features of mechanical glassines at oscillation frequencies 0.1-3 kHz; in particular, the internal friction has a sharp maximum as a function of the frequency. This is the first example of biopolymer glassines at physiological temperatures, because well-known glassy features of DNA and globular proteins are seen only for much lower temperatures (around 200 K). here-concerning, in particular, the specific role of the hydrated water-the rough physical picture of the glass transition in globular proteins is constructed by an analogy with glass-forming liquids [7,8] and synthetic polymers [9,10]. In particular, it is believed that the largescale conformational motion of proteins freezes at ≈ 200 K, analogously to freezing of cooperative motion in glassforming liquids [7,8] and segmental motion in synthetic polymers [9,10].Thus, the glassy features as such are not important in the native state of globular proteins at physiological temperatures, though the glass transition at much lower temperatures allows to gain some understanding of relevant motions in proteins [7,8].Here we shall demonstrate via micro-mechanical methods that the native type I collagen fibril (made of fibrous protein, type I collagen triple-helices) is in a glassy state at physiological temperatures. This state is displayed via frequency-dependent visco-elastic characteristics (the Young's modulus and the damping decrement) of the native fibril. Upon heating the fibril goes out of the glassy state, a phenomenon known as the softening transition [9,10]. The temperature of this transition depends essentially on the speed of heating, e.g., for the standard heating speed v = 1 C/min it occurs around ≈ 70 C. Note that around 70 C the fibril starts to undergo the denaturation process [13][14][15][16]. This process has been studied by different methods, and some of those methods seems to indicate that this process is inherently irreversible [14]. We saw that upon decreasing the heating speed to v = 0.1 C/min, the softening transition takes place at ≈ 25 C, showing that the glassy features pertain to the native state of the collagen fibril and do not directly relate to its denaturation. We shall also confirm that the glassy features are not seen for the heat-denaturated collagen fibril, and contrast features of the native collagen fibril to those of globular proteins.Type I collagen is the major structural element in the extra-cellular matrix. It forms the basis of fibrous connective tissues, such as tendon, chord, skin, bones, corn...

At 49 • C erythrocytes undergo morphological changes due to an internal force, but the origin of the force that drives changes is not clear. Here we point out that our recent experiments on thermally induced force-release in hemoglobin can provide an explanation for the morphological changes of erythrocytes.