The ability of salts to stabilize proteins in vivo or intracellularly correlates with the Hofmeister series of ions.

Numerous studies have been conducted on the ability of salts to stabilize proteins in vitro using purified proteins demonstrating the fact that the ability of salts to stabilize proteins correlates with the Hofmeister series of ions. Using the well characterized bacterial aqueous cytosolic ß-galactosidase and catechol 2,3-dioxygenase enzymes, we demonstrated that salts can stabilize proteins in vivo or intracellularly as well and that the ability of salts to stabilize these two proteins intracellularly also correlates with the Hofmeister series of ions. Na2SO4 and Na2HPO4 were very effective at stabilizing both proteins, followed by NaCl, NH4Cl and (NH4)2HPO4, while NH4CH3CO2, (NH4)2SO4 and NaCH3CO2 did not stabilize either of the proteins. We also investigated the ability of salts to rescue a collection of well characterized nonfunctional ß-galactosidase and catechol 2,3-dioxygenase missense mutants that our laboratory has created. 73.33% of the ß-galactosidase missense mutants could be rescued by salt, while only 33.33% of the catechol 2,3 dioxygenase missense mutants could be rescued by salt. This observation was explained by the differences in densities for the two proteins. Catechol 2,3 dioxygenase is almost twice as dense or compact as ß-galactosidase and thus it is far easier for salts to penetrate and rescue inactive ß-galactosidase proteins. 68.42% of the missense mutants that were rescuable by salt contained mutations that affected amino acids on the surface of the protein and is consistent with the likelihood that salt is able to rescue missense mutants that affect amino acids located on the surface of the protein much more readily than salt can rescue missense mutants that affect amino acids buried in the protein.

Nonfunctional Missense Mutants in Two Well Characterized Cytosolic Enzymes Reveal Important Information About Protein Structure and Function.

The isolation and characterization of 42 unique nonfunctional missense mutants in the bacterial cytosolic ß-galactosidase and catechol 2,3-dioxygenase enzymes allowed us to examine some of the basic general trends regarding protein structure and function. A total of 6 out of the 42, or 14.29% of the missense mutants were in α-helices, 17 out of the 42, or 40.48%, of the missense mutants were in ß-sheets and 19 out of the 42, or 45.24% of the missense mutants were in unstructured coil, turn or loop regions. While α-helices and ß-sheets are undeniably important in protein structure, our results clearly indicate that the unstructured regions are just as important. A total of 21 out of the 42, or 50.00% of the missense mutants caused either amino acids located on the surface of the protein to shift from hydrophilic to hydrophobic or buried amino acids to shift from hydrophobic to hydrophilic and resulted in drastic changes in hydropathy that would not be preferable. There was generally good consensus amongst the widely used algorithms, Chou-Fasman, GOR, Qian-Sejnowski, JPred, PSIPRED, Porter and SPIDER, in their ability to predict the presence of the secondary structures that were affected by the missense mutants and most of the algorithms predicted that the majority of the 42 inactive missense mutants would impact the α-helical and ß-sheet secondary structures or the unstructured coil, turn or loop regions that they altered.

The Promiscuous sumA Missense Suppressor from Salmonella enterica Has an Intriguing Mechanism of Action.

While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.