Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12 - 16, 11000 Belgrade, Serbia.
Biochimie. 2012 Feb;94(2):510-5. doi: 10.1016/j.biochi.2011.08.020. Epub 2011 Sep 2.
Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. (31)P NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.
了解表面电荷对蛋白质稳定性的影响是“定制”具有增加热稳定性的蛋白质的前提条件之一。在这里，我们研究了最近分离的四种外切蔗糖酶同工酶（EINV1-EINV4）之间观察到的热稳定性变化的起源。来自酵母酿酒酵母的外切蔗糖酶是一种同源二聚体糖蛋白，它是研究糖基成分对蛋白质稳定性影响的广泛使用的模型。四种同工酶的稳定性从 EINV1 到 EINV4 降低，同时负表面电荷密度增加。质谱分析表明，同工酶具有相同的蛋白质部分，表明稳定性的差异是翻译后修饰的结果。（31）P NMR 分析表明，同工酶含有以二酯和单酯形式连接到聚糖部分的带负电荷的磷酸基团。不同同工酶之间结合的磷酸总量不同。这些结果以及对多甘露聚糖成分数量的分析表明，负表面电荷密度并不完全取决于磷酸的数量，而是取决于其分布。这表明结合在糖蛋白糖基部分的带电基团可以影响糖蛋白的稳定性。
