To Hofmeister and beyond: an improved understanding of protein solubility and stability

蛋白质在药物和诊断配方、化妆品、工业过程和洗涤剂中的应用越来越广泛。一个主要问题是，蛋白质在液体配方中的溶解度和/或稳定性有限，随着时间的推移，导致质量下降，效益降低，成本浪费（特别是蛋白质通常是最昂贵的部分），以及缺乏可重复性和可预测性。类似的问题也存在于其他领域：例如，许多蛋白质的分析光谱很差，在饮料中呈浑浊或雾状，由于在溶液中保持它们很困难，因此无法结晶。因此，主要需要在不失去溶解度的情况下提高稳定性，反之亦然。使用的主要方法是向蛋白质溶液中添加高浓度的离子和其他分子（通常称为辅料）。目前，辅料的选择通常是在特定的基础上进行的，使用不同溶液条件的高通量测试，并且很少有理论依据可以作为更合理，更便宜和更快提高性能的途径的基础。该建议旨在为理解和提高稳定性和溶解度提供更合理的依据。我们最近提出了一个模型，解释了添加到蛋白质溶液中的盐如何通过与蛋白质竞争水分子来影响其溶解度和稳定性。不同的盐与蛋白质的竞争有好有坏，因此有不同的效果。这种效应在100多年前就已为人所知，通常被称为霍夫迈斯特效应，但对其解释仍存在相当大的分歧。这很重要，因为一旦我们了解了霍夫迈斯特效应的物理基础，我们就可以利用它来开发更好的溶液条件。我们的模型在很大程度上是基于一种被称为核磁共振的光谱技术的测量。该模型与当前的标准模型在许多重要方面有所不同。因此，我们将首先使用NMR将我们的模型与其中最流行的模型进行比较，以消除对我们模型最可能存在的反对意见并清理基础。我们的模型强调了水分子在调节溶解度和稳定性变化中的作用：特别是，它预测了稳定蛋白质的离子必然会降低蛋白质的可溶性，反之亦然。如果这是严格正确的，那么它就严重限制了我们在不影响溶解度的情况下提高稳定性所能做的事情。因此，我们要设法绕过这个问题。首先，我们将研究离子的混合物是否以简单的加性方式表现。在某种程度上，如果他们不这样做，我们或许能够利用一个机会之窗。特别有趣的一对离子是精氨酸和谷氨酸两种氨基酸的混合物，它们似乎以不同的方式起作用，显然是“特殊的”。我们将研究它们是如何工作的，它们实际上是否不同，以及将它们与更典型的霍夫迈斯特离子结合是否有帮助。我们还将调查我们的怀疑，即增加蛋白质的大小会使添加的离子的作用减弱：如果这是真的，这是有用的信息，因为它指导了我们如何稳定不同的蛋白质。有许多生物可以在高盐浓度下生长。通常，高盐会降低溶解度或稳定性，或者两者兼而有之：那么这些生物是如何成功生长的呢？答案是它们产生高浓度的特定小分子来平衡高的外部离子强度。我们将研究这些分子是如何工作的，以及它们的行为是否与“正常”离子相同。如果它们以不同的方式起作用，那么它们就可以被利用。最后，我们将研究外部压力是否可以作为稳定蛋白质的另一种方法，从而研究压力是否会是一个有用的变量。结果将是一个合理的工具箱来指导解决方案的条件。

Proteins are used increasingly widely, in pharmaceutical and diagnostic formulations, cosmetics, industrial processes, and detergents. A major problem is that proteins have limited solubility and/or stability in liquid formulations, leading over time to loss of quality, reduced benefit, wasted cost (especially as the protein is usually the most expensive part), and a lack of reproducibility and predictability. Similar problems abound in other spheres: for example many proteins give poor analytical spectra, cloudiness or haze in beverages, and cannot be crystallised, because of difficulties in keeping them in solution. There is thus a major need to improve stability without losing solubility or vice versa. The main approach used is to add high concentrations of ions and other molecules to the protein solution (known generally as excipients). Currently the selection of excipients is usually tackled on an ad hoc basis, using high-throughput testing of different solution conditions, and there is very little theoretical rationale that could be used as a basis for a more rational, cheaper and quicker route to improved performance. This proposal aims to provide a more rational basis for understanding and improving stability and solubility.We recently proposed a model that explains how salts added to a protein solution affect its solubility and stability, by competing with the protein for water molecules. Different salts compete better or worse than the protein, and therefore have different effects. The effects have been known for over 100 years and are generally called the Hofmeister effect, but there remains considerable disagreement over the explanation. This matters, because once we understand the physical basis for the Hofmeister effect, we can exploit it to develop better solution conditions. Our model was based largely on measurements using a spectroscopic technique known as NMR.The model differs in important ways from the current standard models. We will therefore start by using NMR to compare our model to the most popular of these, to eliminate the most significant likely objection to our model and clear the ground. Our model emphasises the role of water molecules in mediating changes in solubility and stability: in particular, it predicts that ions that stabilise proteins necessarily make them less soluble, and vice versa. If this is strictly true, then it places severe limits on what we can do to (for example) improve stability without compromising solubility. We shall therefore explore ways of getting round the problem. First, we shall investigate whether mixtures of ions behave simply in an additive way. To the extent that they do not, we might be able to exploit a window of opportunity. A particularly interesting pair of ions is a mixture of the two amino acids arginine and glutamate, which seem to work in a different way and are apparently 'special'. We shall investigate how they work, whether they are in fact different, and whether combining them with more typical Hofmeister ions helps. We will also investigate our suspicion that increasing the size of a protein makes the effect of added ions weaker: if true, this is useful information, since it guides the way we would go about stabilising different proteins.There are many organisms that can grow in high salt concentrations. Normally, high salt reduces either solubility or stability or both: so how do these organisms manage to grow successfully? The answer is that they produce high concentrations of specific small molecules to balance the high external ionic strength. We shall investigate how these molecules work, and whether they behave in the same way as 'normal' ions. If they work in different ways, then these can be exploited.Finally we will study whether external pressure can be used as an alternative way to stabilise proteins, and thus whether pressure would be a useful variable. The outcome will be a rational toolbox to guide solution conditions.