Electrostatic interactions modulate the conformation of collagen I
Electrostatic interactions modulate the conformation of collagen I
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DOI：
10.1529/biophysj.106.094284
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发表时间：
2007-03-15
影响因子：
3.4
通讯作者：
Werner, Carsten
中科院分区：
文献类型：
--
作者：
Freudenberg, Uwe;Behrens, Sven H.;Werner, Carsten
利用微缝电动装置，通过流电位/流电流实验，分析了胶原原纤维的pH和电解质依赖性充电。采用差示扫描量热法和圆二色光谱法在相似的电解质溶液中表征静电相互作用对蛋白质构象稳定性的影响。发现胶原I在KCl和CaCl2溶液中的酸碱行为受到离子强度的强烈影响。KCl离子强度从10(-4)M增加到10(-2)M，使蛋白质的等电点（IEP）从pH 7.5增加到5.3。然而，CaCl2溶液中离子强度的类似增加使IEP从7.5上升到pH 9以上。差示扫描量热法观察到两种电解质体系的热稳定性随离子强度的增加而增强。与此相一致的是，圆二色光谱结果表明，螺旋度随着离子强度的增加而增加。更好地筛选带电残基和盐桥的形成被认为是在两种电解质系统中随着离子强度的增加而导致胶原I稳定的原因。氢氧根离子在KCl溶液中优先吸附在本质不带电的位点上，钙离子在CaCl2溶液中与带负电的羧酸基团结合，从而改变了IEP，影响了蛋白质的构象稳定性。
The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl2 solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl2 solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl2 solutions are concluded to shift the IEP and influence the conformational stability of the protein.