Analysis of site-specific histidine protonation in human prolactin.
Analysis of site-specific histidine protonation in human prolactin.
复制标题
人催乳素中位点特异性组氨酸质子化的分析。
DOI：
10.1021/bi800444t
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Hodsdon,MichaelE
中科院分区：
文献类型：
--
作者：
Tettamanzi,MCristina;Keeler,Camille;Meshack,Syrus;Hodsdon,MichaelE
人催乳素（hPRL）是一种23 kDa的蛋白激素和细胞因子，其结构和功能特性依赖于ph值。与hPRL受体胞外结构域结合的解离速率常数在相对狭窄的生理范围内（pH 8 ~ 6）增加了近500倍。由于这种转变的明显中点发生在pH 6.5左右，我们已经将组氨酸残基视为这种行为的潜在生物物理起源。hPRL有9种组氨酸，数量惊人，几乎所有的组氨酸都存在于蛋白质表面。利用核磁共振光谱学，我们监测了这9个残基中的8个位点特异性质子结合，并推导出平衡解离常数。在分析过程中，三种组氨酸（H27， H30和H180）的局部三重态之间的热力学相互作用变得明显，随后通过定点诱变证实了这一点。在考虑了多个电位模型后，我们提出了两个负协同常数的存在，一个是残基H30和H180的连接质子化，其量级约为0.1，另一个是残基H27和H30之间的相互作用较弱。此外，这三种组氨酸中的任何一种突变为丙氨酸，相对于化学变性状态而言，都能稳定折叠后的蛋白质。详细了解这些复杂的质子化反应将有助于阐明ph依赖性调控hPRL结构和功能特性的生物物理机制。
The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.5, we have looked toward histidine residues as a potential biophysical origin of the behavior. hPRL has a surprising number of nine histidines, nearly all of which are present on the protein surface. Using NMR spectroscopy, we have monitored site-specific proton binding to eight of these nine residues and derived equilibrium dissociation constants. During this analysis, a thermodynamic interaction between a localized triplet of three histidines (H27, H30, and H180) became apparent, which was subsequently confirmed by site-directed mutagenesis. After consideration of multiple potential models, we present statistical support for the existence of two negative cooperativity constants, one linking protonation of residues H30 and H180 with a magnitude of approximately 0.1 and the other weaker interaction between residues H27 and H30. Additionally, mutation of any of these three histidines to alanine stabilizes the folded protein relative to the chemically denatured state. A detailed understanding of these complex protonation reactions will aid in elucidating the biophysical mechanism of pH-dependent regulation of hPRL’s structural and functional properties.