Rossi A, Zuccarello L V, Zanaboni G, Monzani E, Dyne K M, Cetta G, Tenni R
Dipartimento di Biochimica Alessandro Castellani, University of Pavia, Italy.
Biochemistry. 1996 May 14;35(19):6048-57. doi: 10.1021/bi9518151.
The properties of type I collagen CNBr peptides in solution were studied to investigate the molecular species formed, their conformation, and factors influencing equilibria between peptide species. Peptides formed homologous trimers, even though the native parent protein is heterotrimeric, [alpha 1(I)]2 alpha 2-(I). Their triple-helical content was found to be high (> 75% for most peptides). Full helical content was not reached mainly because of the presence of monomer species; chain misalignment, if present, and trimer unraveling at terminal ends appeared to play a minor role in reducing helicity. Circular dichroism spectra and resistance to trypsin digestion at 4 and 20 degrees C demonstrated that the conformation of trimers was very similar to the collagen triple-helical conformation. Rotary shadowing of peptide alpha 1(I) CB7 supported this finding. Analytical gel filtration in nondenaturing conditions showed that the trimers of some peptides have the ability to autoaggregate. In the case of peptides alpha 1(I) CB8 and alpha 2(I) CB4, most of the intermolecular interactions between trimeric molecules were disrupted by 0.5 M NaCl, demonstrating that their ionic character is important. Changes in ionic strength also altered the hydrodynamic size of single- and triple-stranded molecules. The different molecular species are in equilibrium. The kinetics of the conversion of trimer to monomer species was determined in a time course experiment using trypsin digestion and found to be a relatively slow process (trimer half-life is a few days at 4 degrees C, about one order of magnitude lower at 20 degrees C) with an activation energy of roughly 4-9 kcal/mol. The circular dichroism profile at increasing temperatures showed that the melting temperature for triple-helical peptides is about 6-10 degrees C lower than that of the parent native type I collagen. The folding of peptides is a spontaneous process (exothermic but with unfavourable entropy change), and the triple-helical conformation originates solely as the result of the collagen sequence because it forms from heat-denatured samples.
研究了I型胶原CNBr肽在溶液中的性质，以探究形成的分子种类、它们的构象以及影响肽种类之间平衡的因素。肽形成了同源三聚体，尽管天然亲本蛋白是异源三聚体[α1(I)]2α2-(I)。发现它们的三螺旋含量很高（大多数肽>75%）。未达到完全螺旋含量主要是因为单体种类的存在；链错配（如果存在）以及末端三聚体解旋在降低螺旋度方面似乎起次要作用。4℃和20℃下的圆二色光谱和对胰蛋白酶消化的抗性表明，三聚体的构象与胶原三螺旋构象非常相似。肽α1(I) CB7的旋转阴影证实了这一发现。非变性条件下的分析凝胶过滤表明，一些肽的三聚体具有自聚集能力。对于肽α1(I) CB8和α2(I) CB4，三聚体分子之间的大多数分子间相互作用被0.5 M NaCl破坏，表明它们的离子特性很重要。离子强度的变化也改变了单链和三链分子的流体力学尺寸。不同的分子种类处于平衡状态。在使用胰蛋白酶消化的时间进程实验中测定了三聚体向单体种类转化的动力学，发现这是一个相对缓慢的过程（三聚体半衰期在4℃时为几天，在20℃时低约一个数量级），活化能约为4 - 9 kcal/mol。温度升高时的圆二色图谱表明，三螺旋肽的解链温度比亲本天然I型胶原低约6 - 10℃。肽的折叠是一个自发过程（放热但熵变不利），三螺旋构象仅源于胶原序列，因为它是由热变性样品形成的。
