Beatrice M. P. Huyghues-Despointes

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI = 3.5) and a basic variant (pI = 10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulombs Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulombs Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge–charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317–15323).

pK Values of Histidine Residues in Ribonuclease Sa: Effect of Salt and Net Charge

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.

Terminal ion pairs stabilize the second β‐hairpin of the B1 domain of protein G

The effects of terminal ion pairs on the stability of a β‐hairpin peptide corresponding to the C‐terminal residues of the B1 domain of protein G were determined using thermal unfolding as monitored by nuclear magnetic resonance and circular dichroism spectroscopy. Molecular dynamics (MD) simulations were also performed to examine the effect of ion pairs on the structures. Eight peptides were studied including the wild type (G41) and the N‐terminal modified sequences that had the first residue deleted (E42), replaced with a Lys (K41), or extended by an additional Gly (G40). Acetylated variants were made to examine the effect of removing the positive N‐terminal charge on β‐hairpin stability. The rank in stability determined experimentally is K41 > E42 ≈ G41 ≈ G40 > Ac‐K41 > Ac‐E42 ≈ Ac‐G41 > Ac‐G40. The Tm of the K41 peptide is 12 °C higher than G41, while the Tm values for the acetylated peptides are less than their unacetylated forms by more than 15 °C. NOE cross‐peaks between side‐chain methylene groups at the N‐ and C‐termini and larger CαH shifts compared to random values are seen for K41. The addition of 20% methanol increases the stability in K41 and G41. The MD studies complement these results by showing that the charged N‐terminus is important to stability. The type of ion pair observed varies with peptide, and when formed the simulations show that the ion pair can prevent fraying of the β‐strands through electrostatic and hydrophobic contacts. Therefore, introducing favorable electrostatic interactions at the N‐ and C‐termini can substantially enhance β‐hairpin stability and help define the structure. Proteins 2006.

NMR study and molecular dynamics simulations of optimized β‐hairpin fragments of protein G

The stability and structure of several β‐hairpin peptide variants derived from the C‐terminus of the B1 domain of protein G were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N‐terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N‐terminal charge–charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the β‐turn conformation. Finally, we show that the effects of combining these single mutations are additive, suggesting that independent stabilizing interactions can be isolated and evaluated in a simple model system. Our results indicate that the structure and stability of this β‐hairpin peptide can be modulated in numerous ways and thus contributes toward a more complete understanding of this important model β‐hairpin as well as to the folding and stability of larger peptides and proteins. Proteins 2007.

Charge–charge interactions are the primary determinants of the pK values of the ionizable groups in Ribonuclease T1 ☆

Coulombs law and a finite difference Poisson-Boltzmann based analysis are used to predict the pK values for 15 ionizable side chains (6 Asp, 6 Glu and 3 His) in ribonuclease T1. These predicted values are compared to the measured pK values to gain insight into the most important factors that influence the pK values of the ionizable groups in proteins. Charge-charge interactions are clearly the most important factor that determines the pK values of most ionizable groups in ribonuclease T1. However, pK values can be shifted by several pK units by the Born self energy associated with burying ionizable groups and by favorable intramolecular hydrogen bonding.