Protein Stabilization and Destabilization by Guanidine Salts

本研究的目的是在热力学和分子水平上定义胍盐与蛋白质在天然状态和未折叠状态下的相互作用，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（C1、SO4-2、SCN-、醋酸盐-）中的胍离子（Gua+）和阴离子对蛋白质结构稳定和展开的单独贡献，并在热力学和分子水平上建立这些离子作用的可加性和补偿规律；定义水分子与蛋白质表面接触并与之极弱相互作用在调节中的作用：1)Gua+离子对蛋白质的不稳定性，以及ii)阴离子对Gua+效应的补偿和加和性。胍盐系统涉及蛋白质与Gua+离子和盐阴离子的同时相互作用。这就发挥了离子之间的可加性和补偿现象，以及与蛋白质表面不同位点相互作用的Gua离子之间预期的补偿。测量胍盐与蛋白质在完整和未折叠二硫裂解状态下的热力学（优先）结合将在几种溶剂ph下的宽浓度范围内（0.5至7.0 M GuaHC1）进行。从这些，相互作用等温线的自由能将产生在整个变性剂浓度范围内的天然状态和未折叠状态的蛋白质。作为温度函数的实验将给出相互作用的焓和熵。然后将优先结合值与蛋白质上的位置占用数据进行比较，这将允许将与蛋白质表面接触的水分子分解为可与助溶剂分子交换的水分子，以及完全排除Gua+盐的位点。这种平衡的建立将允许定义弱相互作用的水在稳定蛋白质结构中的作用。比较Gua盐（从硫酸盐，稳定剂到硫氰酸盐，强变性剂）将允许确定胍离子在不稳定过程中的作用。在不同温度下，通过透析平衡耦合高精度密度法来测量优先相互作用；各种光谱技术将被用来定义蛋白质的转变。该研究解决了通过各种添加材料使蛋白质稳定的问题。在用重组技术（如生物技术）表达和加工新合成的蛋白质时，通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，并保持活性物质的完整。增溶和再折叠过程经常在盐酸胍存在的情况下进行，盐酸胍在高浓度时是一种不稳定剂。另一方面，与之相关的盐，胍硫酸酯是一个很好的稳定剂，但一个较差的增溶剂。目前这类药物的使用大多是经验性的。控制这些试剂作用的规则将通过它们与蛋白质相互作用的详细物理化学研究来建立，水在这些作用中所起的作用将被建立。这些添加剂和溶剂介质中其他添加剂之间的可加性和补偿规则的合理发展，反过来将允许强弱稳定剂和不稳定剂的复杂溶剂混合物的合理配方，系统地用于新合成蛋白质的提取和折叠，以及它们以活性形式的长期储存。* * *

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. ***