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. This is substantially higher than that of ∼ 12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.

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 ß 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. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the ß-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions.

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. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

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. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise.

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. Fourteen Asp and Glu residues titrate with relatively normal pK(a) values that are depressed by less than 1.1 units relative to the normal pK(a) of Asp and Glu in water. Only six residues have pK(a) values shifted by more than 1.5 units. Asp-21 has an unusually high pK(a) of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pK(a) values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pK(a) values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pK(a) values; however, no major pH-sensitive conformational reorganization of the backbone was detected using NMR spectroscopy.

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 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK(a) values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK(a) was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N(epsilon2) state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK(a) calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK(a) values are governed by short-range polar and Coulomb interactions.

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. Each microstate in the native ensemble was modeled to have dual structural character; some regions were treated as folded and retained the same atomic geometry as in the crystallographic structure while other regions were treated thermodynamically as if they were unfolded. Two sets of pK(a) values were used to describe the affinity of each H(+) binding site. One set, calculated with a standard continuum electrostatics method, describes H(+) binding to sites in folded parts of the protein. A second set of pK(a) values, obtained from model compounds in water, was used to describe H(+) binding to sites in unfolded regions. An empirical free energy function, parameterized to reproduce folding thermodynamics measured by differential scanning calorimetry, was used to calculate the probability of each microstate. The effects of pH on the distribution of microstates were determined by the H(+) binding properties of each microstate. The validity of the calculations was established by comparison with a number of different experimental observables.