Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline
Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline
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DOI：
10.1074/jbc.m703991200
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发表时间：
2007-10-12
影响因子：
4.8
通讯作者：
Brodsky, Barbara
中科院分区：
文献类型：
--
作者：
Mohs, Angela;Silva, Teresita;Brodsky, Barbara
化脓性链球菌细胞表面蛋白Scl2含有一个球状N末端结构域和一个胶原样结构域（GlyXaa- X ' aa） 79，形成三螺旋结构域，热稳定性接近哺乳动物胶原。Hyp是动物胶原蛋白中三螺旋稳定性的主要贡献者，但在细菌中不存在，因为细菌缺乏脯氨酸羟化酶。为了探索细菌胶原三螺旋稳定性的基础，在缺乏Hyp的情况下，对重组Scl2蛋白、从Scl2分离的胶原样结构域和一组模拟Scl2高电荷重复（GlyXaa- X ' aa） n序列的肽进行了生物物理研究。在pH值为7时，sc2蛋白的CD光谱、动态光散射和差示扫描量热分析均显示在36℃附近发生了非常剧烈的热转变，表明球形结构域和三螺旋结构域高度协同展开。胰蛋白酶酶切分离的胶原样结构域在相同温度下发生急剧转变，三肽焓为12.5 kJ/ mol。在低pH下，Scl2及其分离的胶原样结构域在中性pH值下表现出明显的不稳定性，在24℃和27℃时发生了两次热转变。在低pH下，Scl2带电的模型肽也出现了类似的不稳定性，不稳定性的程度与GKD三肽单元产生的强烈pH依赖性一致。Scl2蛋白所含的电荷是人纤维形成胶原蛋白的两倍，其静电稳定的程度与Hyp对哺乳动物胶原蛋白稳定性的贡献相似。高焓对Scl2胶原结构域稳定性的贡献支持了在没有Hyp的情况下水合网络的存在。
The Streptococcus pyogenes cell- surface protein Scl2 contains a globular N- terminal domain and a collagen- like domain, ( GlyXaa- X ' aa) 79, which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple- helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple- helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen- like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive ( GlyXaa- X ' aa) n sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 C, indicating a highly cooperative unfolding of both the globular and triple- helix domains. The collagen- like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/ mol of tripeptide. At low pH, Scl2 and its isolated collagen- like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril- forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.