Changes In pKa Research Articles

Sex-dimorphic glucose transporter-2 regulation of cAMP-protein kinase A (PKA) C-alpha pathway activity and phosphorylation in rat hypothalamic primary astrocyte cultures.

Brain astrocyte glycogenolysis is regulated in part by the second messenger adenosine 3'5'-cyclic monophosphate (cAMP). Hypothalamic astrocyte glycogen metabolism shapes glucose counterregulation, under the control of glucose transporter-2 (GLUT2), a plasma membrane glucose carrier and sensor. Hypothalamic astrocyte cAMP is subject to neurotransmitter control, but effects of nutrient cues on this messenger are unclear. Here, an established hypothalamic primary astrocyte culture model and gene knockdown tools were used to investigate the premise that GLUT2 exerts sex-dimorphic regulation of cAMP-protein kinase A (PKA) signalling in these glia. Data show that basal cAMP was elevated in female versus male; GLUT2 gene silencing up-regulated or down-regulated this profile in male versus female. Glucoprivation increased cAMP content in astrocytes of each sex, yet GLUT2 siRNA pretreatment exacerbated (male) or blunted (female) this stimulatory effect. PKA and phosphoPKA levels in glucose-supplied astrocytes were increased (male) or decreased (female) by GLUT2 knockdown. PKA protein was amplified, yet phosphoPKA was refractory to glucose withdrawal in male, while females showed sustained PKA expression alongside diminished phosphoPKA. GLUT2 siRNA pretreatment exacerbated glucoprivic augmentation of PKA content in male but down-regulated both PKA and phosphoPKA proteins in female. Evidence for parallel GLUT2 siRNA-associated changes in cAMP and PKA, albeit in opposing directions in the two sexes, infers that GLUT2 control of hypothalamic astrocyte cAMP-dependent PKA signalling is sex-specific. Data also disclose that in the female, GLUT2 curbs the baseline phosphoPKA/PKA expression ratio but is not involved in glucoprivic suppression of this ratio.

Constitutional adaptation to pKa modulation by remote ester hydrolysis.

The mechanisms through which environmental conditions affect the expression of interconnected species is a key step to comprehending the principles underlying complex chemical processes. In Nature, chemical modifications triggered by the environment have a major impact on the structure and function of biomolecules and regulate different reaction pathways. Yet, minimalistic artificial systems implementing related adaptation behaviours remain barely explored. The hydrolysis of amino acid methyl esters to their corresponding amino acids leads to a drastic change in pKa (ca. 7 and 9, respectively) that protonates the free amino group at physiological conditions. Dynamic covalent libraries (DCvLs) based on amino acid methyl esters and aldehydes respond to such hydrolysis and lead to constitutional adaptation. Each of the libraries studied experiences a DCvL conversion allowing for constituent selection due to the silencing of the zwitterionic amino acids towards imine formation. The selective action of different enzymes on the DCvLs results in states with simplified constitutional distributions and transient chirality. When additional components (i.e., scavengers) that are not affected by hydrolysis are introduced into the dynamic libraries, the amino acid methyl ester hydrolysis induces the up-regulation of the constituents made of these scavenging components. In these systems, the constituent distribution is resolved from a scrambled mixture of imines to a state characterized by the predominance of a single aldimine. Remarkably, although the final libraries display higher "simplexity", the different transient states present an increased complexity that allows for the emergence of organized structures [micelle formation] and distributions [up-regulation of two antagonistic constituents]. This reactive site inhibition by a remote chemical modification resembles the scenario found in some enzymes for the regulation of their activity through proximal post-translational modifications.

Stretching vibrational frequencies and pKa differences in H-bond networks of protein environments

The experimentally measured stretching vibrational frequencies of O–D [νO–D(donor)] and C=O [νC=O(donor)] H-bond donor groups can provide valuable information about the H-bonds in proteins. Here, using a quantum mechanical/molecular mechanical approach, the relationship between these vibrational frequencies and the difference in pKa values between H-bond donor and acceptor groups [ΔpKa(donor … acceptor)] in bacteriorhodopsin and photoactive yellow protein environments was investigated. The results show that νO–D(donor) is correlated with ΔpKa(donor … acceptor), regardless of the specific protein environment. νC=O(donor) is also correlated with ΔpKa(donor … acceptor), although the correlation is weak because the C=O bond does not have a proton. Importantly, the shifts in νO–D(donor) and νC=O(donor) are not caused by changes in pKa(donor) alone, but rather by changes in ΔpKa(donor … acceptor). Specifically, a decrease in ΔpKa(donor … acceptor) can lead to proton release from the H-bond donor group toward the acceptor group, resulting in shifts in the vibrational frequencies of the protein environment. These findings suggest that changes in the stretching vibrational frequencies, in particular νO–D(donor), can be used to monitor proton transfer in protein environments.

Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

IONIZATION OF ETHANOLAMINES IN WATER- ORGANIC MEDIA

In this paper, the ionization constants (pKa) of some nitrogen-containing organic bases (monoethanolamine, diethanolamine and triethanolamine) in water-ethanol, water-acetone and water-acetonitrile solutions at various concentrations of organic solvent in the studied systems have been determined by the potentiometric titration method. It was found that the values of pKa of monoethanolamine, diethanolamine and triethanolamine in aqueous-organic solutions naturally decreases with increasing concentration of organic solvent in the system. It was shown that their nature and the physico-chemical properties of the medium are significantly influenced by the nature and degree of change in the electron donor ability of the studied nitrogen-containing organic bases. It should be noted that for the studied nitrogen-containing organic bases (monoethanolamine, diethanolamine and triethanolamine), when passing from an aqueous medium to an organic one (ethanol or acetone or acetonitrile), the pKa values naturally decrease with an increase in the content of the organic solvent in the system. In general, in the transition from an aqueous medium to an organic one (ethanol or acetone or acetonitrile), a change in pKa can reach 1.73 units. It was established that the dependence of the values of pKa of nitrogen-containing organic bases which were determinate via potentiometric titration method on the inverse of the dielectric constant of water-ethanol and water-acetone or water-acetonitrile solutions tends to be linear, consistent with Izmailov’s theory. Thus, we can conclude that the change in the ratio of the mixed solvent components and, as a result, in general, dielectric permeability of the medium does not lead to a significant change in the solvation characteristics of the studied nitrogen-containing organic bases.

Computational investigation of explicit solvent effects and specific interactions of hydroxypyrene photoacids in acetone, DMSO, and water.

This work employs the correlated wavefunction-based methods ADC(2) and CC2 in combination with the implicit solvent model COSMO to calculate the UV/Vis absorption and fluorescence emission energies of particularly strong hydroxypyrene photoacids in acetone. According to the Förster cycle, the electronic transition energies are first used to compute , i.e., the pKa change upon excitation and then the excited-state pKa (labeled ) with ground-state pKa values based on COSMO-RS as additional inputs. Furthermore, for the strongest photoacid of that class, namely tris(1,1,1,3,3,3-hexafluoropropan-2-yl)-8-hydroxypyrene-1,3,6-trisulfonate, the need to go beyond implicit solvation and to account for explicit solvent effects on the electronic transition energies and the resulting ΔpKa is investigated in the solvents acetone, dimethyl sulfoxide (DMSO), and water. For this, a hybrid implicit-explicit approach is followed by comparing micro-solvated structures that are generated based on Kamlet-Taft considerations. While implicit solvent effects are mostly sufficient for the aprotic solvent acetone, one explicit solvent molecule seems relevant for DMSO due to its stronger hydrogen-bond (HB) acceptance and hence larger interaction with the photoacid OH group as a HB donor. For the protic solvent water, the situation is more complicated, involving at least one water molecule at the OH group and up to three water molecules at the O- group of the corresponding base. Finally, these results are used to rationalize the experimentally observed spectral evolution of the photoacid absorption band in acetone-water solvent mixtures.

Potential Application of Discarded Natural Coal Gangue for the Removal of Tetracycline Hydrochloride (TC) from an Aqueous Solution

The generation and accumulation of discarded coal gangue (CG) have severe environmental impacts. CG can adsorb other pollutants in the aquatic environment. However, previous studies have not assessed whether CG can adsorb the emerging contaminant tetracycline hydrochloride (TC). Here, discarded CG taken from a mine was pretreated by crushing, cleaning, and sieving and subsequently applied to the adsorption of TC. The adsorption studies were carried out by batch equilibrium adsorption experiments. Our findings indicated that the adsorption behavior could be accurately described using the quasi-first order kinetic and Langmuir adsorption isotherm models, indicating that monolayer adsorption was the main mechanism mediating the interaction between CG and TC. The adsorption process was classified as a thermodynamic endothermic and spontaneous reaction, which was controlled by chemical and physical adsorption, including electrostatic interaction and cation exchange. The pH of the solution had a great influence on the TC adsorption capacity of GC, with higher adsorption occurring in acidic environments compared to alkaline environments. This was attributed to the changes in CG Zeta potential and TC pKa at different pH conditions. Collectively, our findings demonstrated the potential applicability of discarded CG for the adsorption of TC and provided insights into the adsorption mechanisms.

11B NMR Spectroscopy: Structural Analysis of the Acidity and Reactivity of Phenyl Boronic Acid-Diol Condensations.

Phenyl boronic acids are valuable for medical diagnostics and biochemistry studies due to their ability to readily bind with carbohydrates in water. Incorporated in carbohydrates are 1,2-diols, which react with boronic acids through a reversible covalent condensation pathway. A wide variety of boronic acids have been employed for diol binding with differing substitution of the phenyl ring, with the goals of simplifying their synthesis and altering their thermodynamics of complexation. One method for monitoring their pKa's and binding is 11B NMR spectroscopy. Herein, we report a comprehensive study employing 11B NMR spectroscopy to determine the pKa of the most commonly used phenyl boronic acids and their binding with catechol or d,l-hydrobenzoin as prototypical diols. The chemical shift of the boronic acid transforming into the boronate ester was monitored at pHs ranging from 2 to 10. With each boronic acid, the results confirm (1) the necessity to use pHs above their pKa's to induce complexation, (2) that the pKa's change in the presence of diols, and (3) that 11B NMR spectroscopy is a particularly convenient tool for monitoring these interconnected acidity and binding phenomena.

AMOEBA Force Field Trajectories Improve Predictions of Accurate pKa Values of the GFP Fluorophore: The Importance of Polarizability and Water Interactions.

Precisely quantifying the magnitude, direction, and biological functions of electric fields in proteins has long been an outstanding challenge in the field. The most widely implemented experimental method to measure such electric fields at a particular residue in a protein has been through changes in pKa of titratable residues. While many computational strategies exist to predict these values, it has been difficult to do this accurately or connect predicted results to key structural or mechanistic features of the molecule. Here, we used experimentally determined pKa values of the fluorophore in superfolder green fluorescent protein (GFP) with amino acid mutations made at position Thr 203 to evaluate the pKa prediction ability of molecular dynamics (MD) simulations using a polarizable force field, AMOEBA. Structure ensembles from AMOEBA were used to calculate pKa values of the GFP fluorophore. The calculated pKa values were then compared to trajectories using a conventional fixed charge force field (Amber03 ff). We found that the position of water molecules included in the pKa calculation had opposite effects on the pKa values between the trajectories from AMOEBA and Amber03 force fields. In AMOEBA trajectories, the inclusion of water molecules within 35 Å of the fluorophore decreased the difference between the predicted and experimental values, resulting in calculated pKa values that were within an average of 0.8 pKa unit from the experimental results. On the other hand, in Amber03 trajectories, including water molecules that were more than 5 Å from the fluorophore increased the differences between the calculated and experimental pKa values. The inaccuracy of pKa predictions determined from Amber03 trajectories was caused by a significant stabilization of the deprotonated chromophore's free energy compared to the result in AMOEBA. We rationalize the cutoffs for explicit water molecules when calculating pKa to better predict the electrostatic environment surrounding the fluorophore buried in GFP. We discuss how the results from this work will assist the prospective prediction of pKa values or other electrostatic effects in a wide variety of folded proteins.

Theoretical Study on the Photoacidity of Hydroxypyrene Derivatives in DMSO Using ADC(2) and CC2.

This work applies the thermodynamic Förster cycle to theoretically investigate the pKa*, i.e., excited-state pKa values of pyranine-derived superphotoacids developed by Jung and co-workers. The latter photoacids are strong enough to transfer a proton to the aprotic solvent dimethyl sulfoxide (DMSO). The Förster cycle provides access to pKa* via the ground-state pKa and the electronic excitation energies. We use the conductor-like screening model for real solvents (COSMO-RS) to compute the ground-state pKa and the correlated wavefunction-based methods ADC(2) and CC2 with the continuum solvation model COSMO to calculate the pKa change upon excitation. A comparison of the calculated UV/Vis absorption and fluorescence emission energies to the experimental results leads us to infer that this approach allows for a proper description of the electronic excitations. In particular, implicit solvation by means of the COSMO model appears to be sufficient for the treatment of these photoacids in DMSO. The calculations confirm the presumption that a charge redistribution from the hydroxy group to the aromatic ring and the electron-withdrawing substituents is the origin of photoacidity for these photoacids. Moreover, the calculations with the continuum solvation model predict that the pKa jump upon excitation decreases with increasing solvent polarity, as rationalized based on the Förster cycle.

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments.

Labeled ammonium cations with pK a∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pK a 9.8. We report here that primary ammonium cations with higher pK a 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pK a 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self‐assembled into micelles, guanidinium cations enter cells like arginine‐rich cell‐penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pK a change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.

Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes.

Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pKa have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.

Beating Heart of Cytochrome c Oxidase: The Shared Proton of Heme a3 Propionates.

Cytochrome c oxidase (CcO) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through CcO is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in CcO were suggested as PLS candidates although, according to CcO crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a3 propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a. The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the pKA values of all titratable residues in CcO. For the modeled structures, the heme a3 propionates have pKA values high enough to host a proton transiently but not too high to fix the proton permanently. The change in pKA throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.

Determination of pKa and Degradation Products of the Mutated BRAF Inhibitor Vemurafenib and UPLC Assay in Human Urine

In this study, the pKa value of vemurafenib was determined by the dependence of the retention factor on the pH of the mobile phase. The change of pKa in the different acetonitrile fraction ranging between 57.5 and 62.5% (v/v) was studied by using an LC-UV method. Additionally, a simple, reliable, and rapid UPLC method has been developed for the determination of vemurafenib in human urine. Efavirenz was used as an internal standart. For vemurafenib and efavirenz, selectivity factors were found as 1.204. The asymmetry and capacity factors were obtained as 1.100 and 7.532 for vemurafenib.&#x0D; Vemurafenib was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were detected by LC/MS/MS method.

Shifts in the pKa value of acid–base indicators caused by immobilization on solid substrates via water-soluble polycationic polymers: a case study of Congo Red

Objectives. Herein, the effects of cationic polyelectrolytes on the properties of solid substrate immobilized acid–base indicators are investigated to predict shifts in their spectral patterns and characteristics. Methods. The properties of the silica gel immobilized indicator dye in a solution of the cationic polyelectrolyte were studied using automatic photometric titration in the visible region and spectrophotometry using a specialized computerized setup. Results. The measured pKa value of the immobilized dye, which had shifted by three units to the acidic region, was very similar to the pKa value observed for the indicator in the modifying polymer solution. The observed change in pKa of the immobilized dye and the influence of the solution’s ionic strength were attributed to the local electric potential of the polymer globule. In contrast to the processes associated with covalent immobilization, the effect exerted by the solution’s ionic strength on the indicator reaction diminishes, which, in turn, affects the measured values obtained.Conclusions. The creation of a sensor for continuous visualization of pH levels based on Congo Red immobilized on silica gel was described. Here, a color transition was noted between pH 1 and 4. These materials can be used to monitor metal extraction processes from industrial effluents or to optimize the extraction of valuable actinides. The approach demonstrated in this work can be applied to immobilize other indicators for pH level monitoring purposes or the production of sensors for other analytes.

Amide Bond Bioisosteres: Strategies, Synthesis, and Successes.

The amide functional group plays a key role in the composition of biomolecules, including many clinically approved drugs. Bioisosterism is widely employed in the rational modification of lead compounds, being used to increase potency, enhance selectivity, improve pharmacokinetic properties, eliminate toxicity, and acquire novel chemical space to secure intellectual property. The introduction of a bioisostere leads to structural changes in molecular size, shape, electronic distribution, polarity, pKa, dipole or polarizability, which can be either favorable or detrimental to biological activity. This approach has opened up new avenues in drug design and development resulting in more efficient drug candidates introduced onto the market as well as in the clinical pipeline. Herein, we review the strategic decisions in selecting an amide bioisostere (the why), synthetic routes to each (the how), and success stories of each bioisostere (the implementation) to provide a comprehensive overview of this important toolbox for medicinal chemists.

Large Changes in Protonation of Weak Polyelectrolyte Brushes with Salt Concentration-Implications for Protein Immobilization.

We report for the first time that the protonation behavior of weak polyelectrolyte brushes depends very strongly on ionic strength. The pKa changes by one pH step per order of magnitude in salt concentration. For low salt concentrations (∼1 mM), a very high pH is required to deprotonate a polyacidic brush and a very low pH is required to protonate a polybasic brush. This has major consequences for interactions with other macromolecules, as the brushes are actually almost fully neutral when believed to be charged. We propose that many previous studies on electrostatic interactions between polyelectrolytes and proteins have, in fact, looked at other types of intermolecular forces, in particular, hydrophobic interactions and hydrogen bonds.

IONIZATION OF SOME NITROGEN-CONTAINING ORGANIC BASES IN WATER-ETHANOL AND WATER-ACETONE MEDIA

In this paper, the ionization constants (pKa) of some nitrogen-containing organic bases (morfoline, piperidine and benzylamine) in water-ethanol and water-acetone media at various concentrations of organic solvent in the system has been determined by the potentiometric titration method. It was established that the value of pKa of nitrogen-containing organic bases in aqueous-organic media naturally decreases with increasing content of organic solvent in the system. It was shown that their nature (aromatic or heterocyclic) and the physico-chemical properties of the medium are significantly influenced by the nature and degree of change in the electron donor ability of the nitrogen-containing organic bases studied It should be noted that for the studied nitrogen-containing organic bases (morfoline, piperidine and benzylamine), when passing from an aqueous medium to an organic one (ethanol or acetone), the pKa values naturally decrease with an increase in the content of the organic solvent in the system. In general, in the transition from an aqueous medium to an organic one (ethanol or acetone), a change in pKa can reach 2.43 units. It was established that the dependence of the values of pKa of nitrogen-containing organic bases which were determinate via potentiometric titration method on the inverse of the dielectric constant of water-ethanol and water-acetone solutions tends to be linear, consistent with Izmailov’s theory. Thus, we can conclude that the change in the ratio of the mixed solvent components and, as a result, in general, dielectric permeability of the medium does not lead to a significant change in the solvation characteristics of the nitrogen-containing organic bases studied.

Fully Atomistic Multiscale Approach for pKa Prediction.

The ionization state of titratable amino acids strongly affects proteins structure and functioning in a large number of biological processes. It is therefore essential to be able to characterize the pKa of ionizable groups inside proteins and to understand its microscopic determinants in order to gain insights into many functional properties of proteins. A big effort has been devoted to the development of theoretical approaches for the prediction of deprotonation free energies, yet the accurate theoretical/computational calculation of pKa values is recognized as a current challenge. A methodology based on a hybrid quantum/classical approach is here proposed for the computation of deprotonation free energies. The method is applied to calculate the pKa of formic acid, methylammonium, and methanethiol, providing results in good agreement with the corresponding experimental estimates. The pKa is also calculated for aspartic acid and lysine as single residues in solution and for three aspartic/glutamic acids inside a well-characterized protein: hen egg white lysozyme. While for small molecules the method is able to deal with multiple protonation states of all titratable groups, this becomes computationally very expensive for proteins. The calculated pKa values for the single amino acids (except for the zwitterionic aspartic acid) and inside the protein display a systematic shift with respect to the experimental values that suggests that the fine balance between hydrophobic and polar interactions might be not accurately reproduced by the usual classical force-fields, thus affecting the computation of deprotonation free energies. The calculated pKa shifts inside the protein are in good agreement with the corresponding experimental ones (within 1 pKa unit), well reproducing the pKa changes due to the protein environment even in the case of large pKa shifts.

Beyond pKa: Experiments and Simulations of Nitrile Vibrational Probes in Staphylococcal Nuclease Show the Importance of Local Interactions.

Electric fields are fundamentally important to biological phenomena, but are difficult to measure experimentally or predict computationally. Changes in pKa of titratable residues have long been used to report on local electrostatic fields in proteins. Alternatively, nitrile vibrational probes are potentially less disruptive and more direct reporters of local electrostatic field, but quantitative interpretation is clouded by the ability of the nitrile to accept a hydrogen bond. To this end, we incorporated nitrile probes into 10 locations of staphylococcal nuclease (SNase) where pKa shifts had already been determined. We characterized the local environment of each nitrile probe experimentally, through temperature-dependent spectroscopy, and computationally, through molecular dynamics simulations, and show that hydrogen bonding interactions dominate the spectral line shapes. We demonstrate that the information provided by the line shape of the nitrile spectra, compared to scalar values of pKa shift or nitrile frequency shift, better describes local environments in proteins in a manner that will be useful for future computational efforts to predict electrostatics in complex biological systems.