Isom Daniel G, Cannon Brian R, Castañeda Carlos A, Robinson Aaron, García-Moreno Bertrand
Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17784-8. doi: 10.1073/pnas.0805113105. Epub 2008 Nov 12.
Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK(a) values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.
内部可电离基团在水溶性球状蛋白中相当罕见。据推测，这反映了电荷与蛋白质内部疏水环境之间的不相容性。在此我们表明，蛋白质对内部可电离基团可能具有内在的高耐受性。葡萄球菌核酸酶中的25个内部位置一次用赖氨酸、谷氨酸或天冬氨酸进行替换，既不消除酶活性，蛋白质构象也无可检测到的变化。用赖氨酸和谷氨酸对核糖核酸酶H中6个随机选择的内部位置进行替换也得到了类似结果，这表明蛋白质耐受内部可电离基团的能力可能是许多蛋白质共有的特性。所进行的87次替换中有86次使蛋白质稳定性降低，但除一种情况外，所有蛋白质在中性pH下仍保持天然状态。通过比较每种变体蛋白在两个不同pH值下的稳定性，确定大多数内部可电离基团的pK(a)值发生了偏移；许多内部可电离基团在生理pH值下可能呈中性。这些研究表明，可电离基团在蛋白质疏水内部稳定存在不需要特殊的结构适应性。这些研究表明，利用内部可电离基团实现功能目的的酶和其他蛋白质可能是通过随机积累在内部位置引入可电离基团的突变而进化而来，随后经过进化适应和优化来调节稳定性、动力学以及功能所需的其他因素。
