Protein Stabilization and Destabilization by Guanidine Salts

9417773 Timasheff本研究的目的是在热力学和分子水平上确定胍盐与天然和未折叠状态的蛋白质之间的相互作用，从而了解这些相互作用控制球状蛋白质折叠状态的方式;确定胍盐中胍盐和胍盐中阴离子的单独贡献（Cl，SO 4 -2，SCN-，醋酸盐-）蛋白质结构的稳定和展开，并建立这些离子对蛋白质结构的作用的加和和补偿规则。热力学和分子水平;以定义与蛋白质表面接触的水分子的作用，并在i）Gua+离子引起的蛋白质不稳定和ii）阴离子引起的Gua+效应的补偿和加和性的调节中与蛋白质表面非常弱地相互作用。 胍盐体系涉及蛋白质与胍+离子和盐阴离子的同时相互作用。 这就产生了离子之间的加和和补偿现象，以及与蛋白质表面上不同位点相互作用的胍离子之间的预期补偿。 胍盐与处于完整和未折叠二硫键裂解状态的蛋白质的热力学（优先）结合的测量将在几种溶剂pH下在宽浓度范围（0.5至7.0 M GuaHCl）内进行。 由此，对于蛋白质的天然和未折叠状态，在整个变性剂浓度范围内将产生相互作用等温线的自由能。 作为温度函数的实验将给出相互作用的熵和熵。 然后将优先结合值与蛋白质上的位点占用数据进行比较，这将允许将与蛋白质表面接触的水分子分解成可与共溶剂分子交换的水分子和完全排除Gua+盐的位点处的水分子。这种平衡的建立将允许定义弱相互作用水在蛋白质结构稳定中的作用。 瓜盐（从硫酸盐，稳定剂到硫氰酸盐，强变性剂）之间的比较将允许定义胍离子在去稳定化过程中的作用。 优先相互作用将在不同温度下通过透析平衡结合高精度密度测定法进行测量;各种光谱技术将用于定义蛋白质转变。 该研究解决了通过各种添加材料稳定蛋白质的问题。 在通过重组技术表达和加工新合成的蛋白质时，例如在生物技术中使用的，经常需要将它们从细胞中形成的大聚集体（包涵体）中溶解出来，然后将它们折叠成活性结构，并保持该活性材料完整。 增溶和重折叠过程通常在盐酸胍存在下进行，盐酸胍在高浓度下是一种去稳定剂。 另一方面，相关的盐，硫酸胍是一种良好的稳定剂，但是一种不良的增溶剂。 目前，这类药剂的使用大多是经验性的。 通过对这些物质与蛋白质的相互作用进行详细的物理化学研究，将确定这些物质的作用规则，并确定水在这些作用中所起的作用。 这些添加剂和溶剂介质中的其它添加剂之间的加和性和补偿规则的这种合理发展反过来将允许强和弱稳定剂和去稳定剂的复杂溶剂混合物的合理配制，以系统地用于新合成的蛋白质的提取和折叠，以及它们以活性形式的长距离储存。 ***

9417773 Timasheff The objectives of this research is to define on the thermodynamic and molecular levels the interactions between guanidine salts and proteins in the native and unfolded states and, thus, to gain an understanding of the manner in which these interactions control the state of folding of globular proteins; to establish the individual contributions of the guanidinium (Gua+) in and the anions in the Gua+ salts (C1, SO4-2, SCN-, Acetate-) to the stabilization and unfolding of protein structure and to establish the rules of additivity and compensation of the actions of these ions on the thermodynamic and molecular levels; to define the role of water molecules in contact with protein surface and interacting very weakly with it in the modulation of i) protein destabilization by Gua+ ions, and ii) the compensation and additivity of the Gua+ effects by the anions. The system of guanidine salts involves the simultaneous interactions of proteins with the Gua+ ions and the salt anions. This brings into play the phenomena of additivity and compensation between the ions, as well as the expected compensation between Gua ions interacting with different loci on the protein surface. Measurements of the thermodynamic (preferential) binding of guanidine salts with proteins in the intact and unfolded disulfide cleaved states will be carried out over a broad concentration range (0.5 to 7.0 M GuaHC1) at several solvent pHs. From these, free energies of interaction isotherms will be generated over the entire denaturant concentration range for both the native and unfolded states of the protein. Experiments as a function of temperature will give enthalpies and entropies of interaction. The preferential binding values will be compared then with site occupancy data on the protein, which will permit to decompose the water molecules in contact with the protein surface into those that are exchangeable with co-solvent molecules and of those at loci from which the Gua+ salts are totally excluded. The establishment of this balance will permit to define the role of weakly interacting water in the stabilization of protein structure. Comparison between the Gua salts (from sulfate, a stabilizer to thiocyanate, a strong denaturant) will permit to define the role of the guanidinium ion in the destabilization process. The preferential interactions will be measured by dialysis equilibrium coupled with high precision densimetry at various temperature; various spectroscopic techniques will be used to define protein transitions. %%% The research addresses the issue of the stabilization of proteins by various added materials. In the expression and processing of newly synthesized proteins by recombinant techniques, such as used in biotechnology, it is frequently necessary to dissolve them out of large aggregates (inclusion bodies) formed in the cells and then to fold them into an active structure, and to maintain that active material intact. The solubilization and refolding processes are frequently done in the presence of guanidine hydrochloride which, at high concentrations, is a destabilizing agent. On the other hand, the related salt, guanidine sulfate is a good stabilizing agent,but a poor solubilizer. At present the uses of such agents are mostly empirical. The rules that govern the actions of these agents will be established by detailed physical chemical studies of their interactions with proteins and the role played by water in these actions will be established. Such a rational development of the rules of additivity and compensation between these, and other additives to the solvent medium will permit, in turn, the rational formulation of complex solvent mixtures of strong and weak stabilizers and destabilizers to use systematically in the extraction and folding of newly synthesized proteins, as well as in their long range storage in active form. ***