Simons Jan-Willem F A, Kosters Hans A, Visschers Ronald W, de Jongh Harmen H J
Wageningen Centre for Food Sciences, Wageningen, The Netherlands.
Arch Biochem Biophys. 2002 Oct 15;406(2):143-52. doi: 10.1016/s0003-9861(02)00429-0.
Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.
二价钙离子被认为参与分子间蛋白质 - 钙 - 蛋白质交联、分子内静电屏蔽或离子诱导的蛋白质构象变化，作为高温下蛋白质聚集的触发因素。为了研究β-乳球蛋白情况下的前两种现象，采用了化学蛋白质修饰、钙结合和聚集研究相结合的方法，同时保持修饰后蛋白质的结构完整性。尽管通过琥珀酰化增加蛋白质上羧酸盐的数量会导致钙结合能力提高，但钙在诱导蛋白质聚集方面似乎效果较差。事实上，羧酸盐数量越多，触发聚集所需的钙浓度就越高。通过羧酸盐甲基化降低蛋白质表面的负电荷数量会降低钙结合特性，但低浓度下钙诱导的聚集得到改善。单价钠离子不能取代钙的特定作用。净表面电荷与触发聚集所需结合的钙离子数量之间的关系表明，钙需要以阈值亲和力特异性结合羧酸盐位点。随后对表面电荷进行位点特异性筛选会导致蛋白质聚集，这是由蛋白质在高温下的部分展开驱动的，然后由于没有静电排斥而得到促进。
