Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.
Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.
复制标题
通过藻酸盐结合和抑制热聚集来稳定蛋白质。
DOI：
10.1021/acs.biomac.2c00297
复制
发表时间：
2022
影响因子：
6.2
通讯作者：
Leckband,DeborahE
中科院分区：
文献类型：
--
作者：
Chang,Roger;Gruebele,Martin;Leckband,DeborahE
旨在稳定蛋白质的聚合物利用直接相互作用或拥挤，但很少建立增加稳定性或减少聚集的机制。尽管对其对蛋白质稳定性的影响了解有限，但海藻酸盐被广泛用于封装蛋白质以用于药物输送和组织再生。在这里，我们提供的证据表明，海藻酸盐既可以增加蛋白质折叠稳定性，又可以通过直接相互作用而不拥挤来抑制未折叠蛋白质的聚集。我们使用两种蛋白质（代谢蛋白磷酸甘油酸激酶 (PGK) 和 hPin1 WW 结构域）的基于荧光的构象报告基因来监测蛋白质稳定性和聚集，作为温度和溶液中藻酸盐重量百分比的函数。海藻酸盐可使 PGK 稳定高达 14.5 °C，但稳定性高度依赖于蛋白质，而较小的 WW 结构域仅能在 3.5 °C 的温度下稳定以防止热变性。在低藻酸盐重量百分比时稳定性最强，在较高藻酸盐浓度时稳定性降低。这种趋势不能用拥挤来解释，离子筛选表明海藻酸盐通过与重要静电成分的直接相互作用来稳定蛋白质。海藻酸盐还通过与未折叠蛋白质不可逆地结合并防止再折叠来强烈抑制高温下的聚集。海藻酸盐对蛋白质稳定性和聚集的有益和消极影响对实际应用都有重要影响。
Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.