title: pK values of histidine residues in ribonuclease Sa: effect of salt and net charge.
abstract: The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.
authors.affiliation: Department of Medical Biochemistry and Genetics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, TX 77843-1114, USA.
authors.forename: Beatrice M P
authors.initials: BM
authors.lastname: Huyghues-Despointes
authors.forename: Richard L
authors.initials: RL
authors.lastname: Thurlkill
authors.forename: Michael D
authors.initials: MD
authors.lastname: Daily
authors.forename: David
authors.initials: D
authors.lastname: Schell
authors.forename: James M
authors.initials: JM
authors.lastname: Briggs
authors.forename: Jan M
authors.initials: JM
authors.lastname: Antosiewicz
authors.forename: C Nick
authors.initials: CN
authors.lastname: Pace
authors.forename: J Martin
authors.initials: JM
authors.lastname: Scholtz
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D012492:Salts; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000737:chemistry
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D012492:Salts; D014867:Water; D006639:Histidine; D014508:Urea; D012260:Ribonucleases; C077002:ribonuclease Sa3; D008239:Lysine