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.

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein.

An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pKa values near 7. When buried together their pKa values appear to be normal.

Effect of internal cavities on folding rates and routes revealed by real-time pressure-jump NMR spectroscopy.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution.

The pH dependence of staphylococcal nuclease stability is incompatible with a three-state denaturation model.

Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.

Structural, energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity-creating mutations.

The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale.

Remodeling of the folding free energy landscape of staphylococcal nuclease by cavity-creating mutations.

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate.

Structural reorganization triggered by charging of Lys residues in the hydrophobic interior of a protein.

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally.

Cavities determine the pressure unfolding of proteins.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors.

Arginine residues at internal positions in a protein are always charged.

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK(a) values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK(a) values in water.

The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins.

The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins.

Conformational relaxation and water penetration coupled to ionization of internal groups in proteins.

Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged.

Large shifts in pKa values of lysine residues buried inside a protein.

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.

Conformational consequences of ionization of Lys, Asp, and Glu buried at position 66 in staphylococcal nuclease.

The pK(a) values measured previously for the internal Lys-66, Asp-66, and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pK(a) units relative to normal pK(a) values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of the ionizable moieties in crystal structures.

Modulation of buried ionizable groups in proteins with engineered surface charge.

Recent work has shown that proteins can tolerate hydrophobic-to-ionizable-residue mutations. Here, we provide experimental evidence that the essential properties (pK value, protonation state, local dynamics) of buried ionizable groups in proteins can be efficiently modulated through the rational design of the surface charge distribution, thus paving the way for the protein engineering exploitation of charge burial.

Adaptations of proteins to cellular and subcellular pH.

Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.

Relationship between protein stabilization and protein rigidification induced by mannosylglycerate.

Understanding protein stabilization by small organic compounds is a topic of great practical importance. The effect of mannosylglycerate, a charged compatible solute typical of thermophilic microorganisms, on a variant of staphylococcal nuclease was investigated using several NMR spectroscopy methods. No structural changes were apparent from the chemical shifts of amide protons.

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.

High tolerance for ionizable residues in the hydrophobic interior of proteins.

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups.

Towards a quantitative understanding of protein hydration and volumetric properties.

Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Delta+PHS. The temperature-dependent volumetric properties of the folded and unfolded states of the wild-type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level.

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.

Direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein.

We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear 1H-15N NMR data on the Lys-66 side chain amine (Nzeta) group in the delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nzeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hzeta and 15Nzeta chemical shifts at pH 8.

A buried lysine that titrates with a normal pKa: role of conformational flexibility at the protein-water interface as a determinant of pKa values.

Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pK(a) values shifted by up to 5 pK(a) units in the direction that promotes the neutral state. In contrast, the internal Lys-38 in SNase titrates with a normal pK(a). The crystal structure of the L38K variant shows that the side chain of Lys-38 is buried.

Ligand effects on the protein ensemble: unifying the descriptions of ligand binding, local conformational fluctuations, and protein stability.

Detailed description of the structural and physical basis of allostery, cooperativity, and other manifestations of long-range communication between binding sites in proteins remains elusive. Here we describe an ensemble-based structural-thermodynamic model capable of treating explicitly the coupling between ligand binding reactions, local fluctuations in structure, and global conformational transitions. The H(+) binding reactions of staphylococcal nuclease and the effects of pH on its stability were used to illustrate the properties of proteins that can be described quantitatively with this model.