Xu Yujia
Department of Chemistry, Hunter College-CUNY, New York, USA.
Methods Enzymol. 2009;466:211-32. doi: 10.1016/S0076-6879(09)66009-2. Epub 2009 Nov 13.
Chief among the challenges of characterizing the thermal stability of the collagen triple helix are the lack of the reversibility of the thermal transition and the presence of multiple folding-unfolding steps during the thermal transition which rarely follows the simple two-state, all-or-none mechanism. Despite of the difficulties inherited in the quantitative depiction of the thermal transition of collagen, biophysical studies combined with proteolysis and mutagenesis approaches using full-chain collagens, short synthetic peptides, and recombinant collagen fragments have revealed molecular features of the thermal unfolding of the subdomains of collagen and led to a better understanding of the diverse biological functions of this versatile protein. The subdomain of collagen generally refers to a segment of the long, rope-like triple helical molecule that can unfold cooperatively as an independent unit whose properties (their size, location, and thermal stability) are considered essential for the molecular recognition during the self-assembly of collagen and during the interactions of collagen with other macromolecules. While the unfolding of segments of the triple helix at temperatures below the apparent melting temperature of the molecule has been used to interpret much of the features of the thermal unfolding of full-chain collagens, the thermal studies of short, synthetic peptides have firmly established the molecular basis of the subdomains by clearly demonstrating the close dependence of the thermal stability of a triple helix on the constituent amino acid residues at the X and the Y positions of the characteristic Gly-X-Y repeating sequence patterns of the triple helix. Studies using recombinant collagen fragments further revealed that in the context of the long, linear molecule, the stability of a segment of the triple helix is also modulated by long-range impact of the local interactions such as the interchain salt bridges. Together, the combined approaches represent a unique example on delineating molecular properties of a protein under suboptimal conditions. The related knowledge is likely not to be limited to the applications of collagen studies, but contributes to the understanding of the molecular properties and functions of protein in general.
表征胶原蛋白三螺旋热稳定性面临的主要挑战包括热转变缺乏可逆性，以及热转变过程中存在多个折叠-去折叠步骤，而这很少遵循简单的两态、全或无机制。尽管在定量描述胶原蛋白的热转变方面存在困难，但结合蛋白水解和诱变方法的生物物理研究，使用全长胶原蛋白、短合成肽和重组胶原蛋白片段，揭示了胶原蛋白亚结构域热去折叠的分子特征，并有助于更好地理解这种多功能蛋白质的多种生物学功能。胶原蛋白的亚结构域通常指的是长绳状三螺旋分子的一段，它可以作为一个独立单元协同展开，其性质（大小、位置和热稳定性）被认为对于胶原蛋白自组装过程中的分子识别以及胶原蛋白与其他大分子相互作用至关重要。虽然在低于分子表观解链温度的温度下三螺旋片段的去折叠已被用于解释全长胶原蛋白热去折叠的许多特征，但短合成肽的热研究通过清楚地证明三螺旋的热稳定性对三螺旋特征性Gly-X-Y重复序列模式中X和Y位置的组成氨基酸残基的紧密依赖性，牢固地确立了亚结构域的分子基础。使用重组胶原蛋白片段的研究进一步表明，在长线性分子的背景下，三螺旋片段的稳定性也受到局部相互作用（如链间盐桥）的远程影响的调节。总之，这些综合方法是在次优条件下描绘蛋白质分子特性的一个独特例子。相关知识可能不仅限于胶原蛋白研究的应用，而且有助于一般地理解蛋白质的分子特性和功能。
