title: The effect of net charge on the solubility, activity, and stability of ribonuclease Sa.
abstract: The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.
authors.affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Shaw
authors.forename: G R
authors.initials: GR
authors.lastname: Grimsley
authors.forename: G I
authors.initials: GI
authors.lastname: Yakovlev
authors.forename: A A
authors.initials: AA
authors.lastname: Makarov
authors.forename: C N
authors.initials: CN
authors.lastname: Pace
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry*; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics
chemical_list: D007527:Isoenzymes; D001224:Aspartic Acid; D018698:Glutamic Acid; D012260:Ribonucleases; C077002:ribonuclease Sa3; D008239:Lysine