Arginine: Its pKa value revisited.

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1.

Molecular determinants of the pKa values of Asp and Glu residues in staphylococcal nuclease.

Prior computational studies of the acid-unfolding behavior of staphylococcal nuclease (SNase) suggest that the pK(a) values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pK(a) values of carboxylic groups in SNase, the pK(a) values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups.

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease.

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK(a) values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK(a) values of 5.

Structure-based pKa calculations using continuum electrostatics methods.

Electrostatic free energy is useful for correlating structure with function in proteins in which ionizable groups play essential functional roles. To this end, the pK(a) values of ionizable groups must be known and their molecular determinants must be understood. Structure-based calculations of electrostatic energies and pK(a) values are necessary for this purpose.

Electrostatic contributions to the stability of the GCN4 leucine zipper structure.

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK(a) values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK(a) values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p.

Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins.

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized.

High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp.

The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher.

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding.

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.

Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.

Single site mutations that reverse or neutralize a surface charge were made at 22 ionizable residues in staphylococcal nuclease. Unfolding free energies were obtained by guanidine hydrochloride denaturation. These data, in conjunction with previously obtained stabilities of the corresponding alanine mutants, unequivocally show that the dominant contribution to stability for virtually all of the wild-type side chains examined is the electrostatic effect associated with each residue's charged group.

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al.

Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease.

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.

Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein.

Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions.