Study of collagen self-assembly by molecular dynamics and UV spectroscopy techniques.

Collagen is a fibrous protein representing the main constituent of connective tissue in mammals, with a basic structural unit called tropocollagen that is a triple helix consisting of Gly-Xaa-Yaa repetitions, in which one-thirds of the X and Y residues are either prolines or hydroxyprolines. Collagen triple helices associate in fibrils, where tropocollagens are staggered side-by-side with a shift of 67 nm (234 residues) between two neighbours. To investigate the assembly mechanisms, collagen aggregation was studied by means MD simulations both at physiological conditions and at low ionic strength. Two tropocollagen fragments with different hydropathic profiles were chosen and built from Rattus norvegicus type I collagen sequence. Other fragments were selected from the same sequence with a shift of 234 residues upstream and downstream of it. Repeated MD simulations suggest that tropocollagens prefer to associate in pairs, with first approach between hydrophobic regions, suggesting that the mechanism is mainly driven by hydrophobic effect and mediated by hydroxyprolines. Association of two, three or four fragments shows that the amino-acidic composition of the triple helices strongly influences their assembly propensity: poorly charged (PC) segments easily associate at 0.1 M salt concentration, contrary to highly charged (HC) ones. As expected, HC fragments are more suited to self-assembly at low ionic strength. Collagen self-assembly was monitored in vitro by measuring the turbidity changes of the solution as observed from the increase in absorbance at 310 nm. Rat tail tendon collagen was prepared at low temperature and at different pH. Curves of aggregate fractions vs time display a sigmoid profile, indicating, according with literature, a cooperative process with a lag phase whose length depends on the solution pH.