期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2008
卷号：105
期号：46
页码：17784-17788
DOI：10.1073/pnas.0805113105
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 pKa 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.
关键词：electrostatics ; internal ionizable groups ; pKa values ; dehydration
