Structural basis for cyclodextrins' suppression of human growth hormone aggregation
Structural basis for cyclodextrins' suppression of human growth hormone aggregation
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DOI：
10.1110/ps.0202702
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发表时间：
2002-07-01
期刊：
影响因子：
8
通讯作者：
Wimmer, R
中科院分区：
文献类型：
--
作者：
Otzen, DE;Knudsen, BR;Wimmer, R
许多治疗性蛋白质需要在室温下长时间储存。这可能导致聚集和功能丧失。环糊精（CDs）已被证明是多种蛋白质的聚集抑制因子。它们的效力通常归因于它们对芳香氨基酸的亲和力，否则其表面暴露将导致蛋白质结合。然而，没有详细的结构研究。在这里，我们研究了人类生长激素（hGH）在低pH下与不同CDs之间的相互作用。尽管hGH在1 M NaCl中在pH为2.5时很容易聚集形成无定形聚集体，但25至50 mM的各种β - cd衍生物的存在足以完全避免这种情况。-和- cd的效果要差得多。根据极简关联模型分析了β - cd存在下聚集反应的停流数据，得出hGH- β - cd解离常数近似于6 mM。该值与用β - cd对hGH进行简单荧光滴定得到的值非常相似。核磁共振研究表明，β - cd在低pH下导致hGH更不展开的构象，并主要与芳香侧链结合。这表明芳香氨基酸是残馀结构区域的重要组成部分，可能形成聚集核。
Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various beta-CD derivatives is sufficient to completely avoid this. alpha- and gamma-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of beta-CD are analyzed according, to a minimalist association model to yield an apparent hGH-beta-CD dissociation constant of similar to6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with beta-CD. Nuclear magnetic resonance studies indicate that beta-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.