The conformational dynamics of the tetramer hemoglobin molecule as revealed by hydrogen exchange. III. Influence of the heme removal

Two main types of conformational fluctuations--local and global are characteristic of the native protein structure and revealed by hydrogen exchange. The probability of those fluctuations changes to a different extent upon hemoglobin oxygenation, changing of pH, splitting of the intersubunit contacts. To compare with the influence of the heme removal the rate of the H-D exchange of the peptide NH atoms of the human apoHb was studied at the pH range 5.5-9.0 and temperature 10-38 degrees C by the IR spectroscopy. The removal of the heme increases the rate of the H-D exchange of the 80% peptide NH atoms with the factor retardation of the exchange rate (P) in the range approximately 10(2)-10(8). For the most of the peptide NH atoms the probability of the local fluctuations weakly depends on the temperature, the enthalpy changes upon all such local conformational transitions deltaH(op) degrees are 0-15 kcal/M. Characterized by the stronger temperature dependence the global fluctuations are not arised upon the temperature increases up to 38 degrees C at pH 7.0 inspite of in these conditions the slow denaturation and aggregation of apoHb begin to occur. Upon the destabilization of the apoHb structure by the simultaneous decreasing of pH to 5.5 and temperature to 10 degrees C the global fluctuations of the apoHb native structure described by deltaH(op)o < 0 begin to intensify. The mechanism of the overall intensification of the local fluctuations upon the heme removal, the peculiarity of the heat denaturation of apoHb in conditions, close to that existing upon the selfassembly of Hb in vivo, and analogy between low temperature global fluctuations and cold denaturation of globular proteins are discussed.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: 2. Effect of intersubunit contact cleavage

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in isolated α and β subunits of human hemoglobin (Hb) at pH 5.5–9.0 and 20°C. The H-D exchange occurs by the EX2 mechanism. The retardation factor of subunit exchange rate (P) is in a range of approximately 10²–10⁷. Compared to tetrameric Hb, the probability of local fluctuations (1/P) increases to a slightly greater extent in monomeric α subunits than in tetrameric β subunits. Unlike in the whole Hb molecule, oxygenation of its subunits has no effect on the probability of local fluctuations, and the subunits show no pH-dependent changes in 1/P values (observed for liganded Hb). Probable mechanisms accounting for overall intensification of local fluctuations upon the cleavage of contacts between subunits of the tetrameric Hb molecule are discussed with regard to structural crystallographic data.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: I. Effects of pH,temperature, and ligand binding

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in different forms of human hemoglobin (Hb) at pH 5–10 and temperatures of 10–63°C. The pH dependence of the H-D exchange rate fits the EX2 mechanism. At 10–30°C, there are two pH-dependent conformers of liganded Hb forms, the fluctuation probability being lower for the alkaline conformer. The differences between the conformers disappear at 40°C, where a third conformer, with a higher probability of local fluctuations, appears. Deoxyhemoglobin has no pH-dependent conformers in the pH range 6–9 at 20°C, and the probability of local fluctuations is considerably decreased compared to the acid conformer of liganded Hb. The destabilization of the liganded Hb structure by decreasing the pH to 5.0 at 20°C or increasing the temperature to 50–60°C at pH 7.1 enhances global fluctuations of the native structure ensuring the H-D exchange of slowly exchanging NH atoms. The mechanisms of local and high-temperature global fluctuations, as well as the possible similarity between the two pH-dependent conformers of liganded Hb and its functional R and R2 states revealed by X-ray analysis and NMR spectroscopy, are discussed.     

Front Cover Image,Volume 117, Number 1, January 2020

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, −80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and β subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O₂ equilibrium curve, O₂ dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.     

The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. I. Influence pH, temperature and ligand binding

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.     

The mechanisms of the proteolytic degradation of native globular proteins. The role of local and global fluctuations of the native structure

Analysis of the proteolytic degradation of the native protein structure carried out by the comparison of the temperature dependence of the hydrogen exchange and proteolytic splitting rates of the hen egg-white lysozyme and human Hb and apoHb. Acceleration of the burst-like (all or none) proteolytic degradation in the high temperature range is provided by the intensification of the global fluctuations with overall unfolding revealed by hydrogen exchange. For Hb and apoHb the rate of burst-like proteolytic degradation and hydrogen exchange weakly depends on temperature in the range, where hydrogen exchange reveals only local fluctuations of the native protein structure. The splitting of the two proteins proceeds by the selfaccelerated burst-like mechanism with the initial rate-limiting single cleavage owing to the local fluctuation of the native structure. The local fluctuations play important role also upon the intracellular burst-like degradation of native proteins.     

Mechanisms involved in proteolytic degradation of globular proteins: The role of local and global fluctuations in the native structure

The mechanisms involved in degradation of the native protein structure was analyzed by comparing the temperature dependences of the hydrogen exchange (HE) and proteolytic cleavage rates of hen egg lysozyme (HEL), human hemoglobin (Hb), and its apoform (apoHb). Acceleration of the burstlike (all or none) proteolytic degradation of HEL in a high temperature range results from the intensification of global fluctuations with overal structure unfolding, indicated by HE. The rates of Hb and apoHb burstlike degradation and HE weakly depend on the temperature in the range where only local fluctuations of the native structure are detectable by HE. These two proteins are cleaved according to a self-accelerated burstlike mechanism with the initial rate-limiting single cleavage due to local fluctuations in the native structure. Such fluctuations play an important role in intracellular burstlike proteolytic degradation of native proteins.     

Hydrogen exchange and proteolytic degradation of ribonuclease A. Similarities and distinctions of the kinetic mechanisms

The studies by IR spectroscopy of the temperature dependence of the H-D exchange rate of the RNase A peptide NH atoms permit one to characterize two types of conformation fluctuations, local and global. A comparison with the temperature dependence of the proteolytic degradation rate of RNase A shows that similar in nature fluctuations allow for the H-D exchange of NH atoms and the splitting of peptide bonds of the native protein. In the low temperature region, both processes occur through local fluctuations, by way of the EX2 mechanism, and in the high temperature region, they occur through global fluctuations with the overall denaturation desorganization of the native structure, by way of the EX1 mechanism. The biphasic dependence of the rate of H-D exchange and proteolytic degradation of RNase A on urea concentration is also explained by the combination of local and global fluctuations.     

Stability of β-Lactoglobulin A in the Presence of Sugar Osmolytes Estimated from Their Guanidinium Chloride-Induced Transition Curves

The stabilizing role of Trehalose, Sucrose, Sorbitol and Mannitol as sugar osmolytes and polyols on beta-lactoglobulin A (β-lgA) against its chemical denaturation at pH 2.0 and 25 °C has been explored by means of UV–vis spectroscopy. It has been observed that ΔG _D ^o of β-lgA in the presence of 10% (w/v) Trehalose, Sucrose, Sorbitol and Mannitol is increased. We report that the functional dependence of ΔG _D of proteins in the absence and the presence of sugar osmolytes on denaturant concentration is linear. Trehalose is found to induce remarkable stability of β-lgA against chemical denaturation. Furthermore, we assumed sugar osmolytes do not affect the secondary and tertiary structures of the native and GdnHCl-denatured states.     

The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. II. Influence of the intersubunit contact splitting

The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.     

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

The hydrogen–deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H-D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H-D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H-D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H-D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature-jump method. This means that a protein molecule in solution fluctuates between the N- and D-states at every temperature within the transition region, where the N-form is the tightly folded native structure and the D-form the randomly coiled chain. From measurements of thermal unfolding of ester-108-lysozyme and the binding constant of (NAG)₃ to ester-108-lysozyme, it was found that almost all cross-linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H-D exchange reaction of ester-108-lysozyme. In the temperature region of 43–50°C, about 70% of the exchangeable tryptophan residues of ester-108-lysozyme were exchanged within 1 s immediately after the mixing of D₂O, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a corss-linked molecule.     

Bioremediation potential of Chondrus crispus (Basin Head) and Palmaria palmata: effect of temperature and high nitrate on nutrient removal

To evaluate the nutrient removal capabilities of two red macroalgae, apical blades were cultured in the lab for 4?weeks at either 6, 10, or 17°C and nitrate at either 30 or 300?μM, typical of the seasonal range of conditions at a land-based Atlantic halibut farm. Stocking density was 2.0?g?L^?1, irradiance 125?μmol?photons?m^?2?s^?1, photoperiod 16:8 (L:D), and nitrogen to phosphorus ratio 10:1. For both species, the highest growth rate was at 300?μM NO ₃ ^? with Palmaria palmata growing fastest at 6°C, 5.8%?day^?1, and Chondrus crispus growing best at 17°C, 5.5%?day^?1. Nitrogen and carbon removal by P. palmata was inversely related to temperature, the highest rate at 6°C and 300?μM NO ₃ ^? of 0.47?mg N and 6.3?mg C per gram dry weight per day. In contrast, C. crispus removal of N was independent of temperature, with mean removal of 0.49?mgN?gDW^?1?day^?1 at 300?μM NO ₃ ^? . The highest carbon removal by C. crispus was 4.4?mgC?gDW^?1?day^?1 at 10°C and 300?μM nitrate, though not significantly different from either 6 or 17°C and 300?μM nitrate. Tissue carbon:nitrogen ratios were >20 in both species at 30?μM nitrate, and all temperatures indicating nitrogen limitation in these treatments. Phycoerythrin content of P. palmata was independent of temperature, with means of 23.6?mg?gFW^?1 at 300?μM nitrate. In C. crispus, phycoerythrin was different only between 6°C and 17°C at 300?μM nitrate, with the highest phycoerythrin content of 12.6?mg?gFW^?1 at 17°C. Morphological changes were observed in P. palmata at high NO ₃ ^? concentration as curling of the fronds, whilst C. crispus exhibited the formation of bladelets as an effect of high temperature.     

Nitrogen and phosphorus interchange between sediments and overlying water of a wastewater retention pond

Nitrogen and P interchange between the sediments and the overlying water of a simulated retention pond used for wastewater treatment were evaluated under conditions of seasonal temperature fluctuations and varying physico-chemical conditions (exposing floodwater surface to daylight vs. dark and turbulent vs. quiscent floodwater). Natural sediment columns obtained from two types of field retention ponds were used. One type of retention pond consisted of calcareous clay loam sediment while the sediment of second retention pond contained organic soil. Nutrient interchange between sediments and the overlying water was measured once a month over a period of one year. Nitrogen removal rates from floodwater were controlled by the initial floodwater NH ₄ ⁺ and NO ₃ ^? concentration, rate of NH ₄ ⁺ diffusion from the sediments to the overlying water, ammonification in the sediments, NH₃ volatilization and nitrification at the sediment-water interface, and denitrification in the sediments. Under the conditions studied, NH ₄ ⁺ concentrations of the floodwater were in the range of 0.01 to 0.05 µg/ml, while NO ₃ ^? concentrations were in the range of 0.27 to 0.78 µg/ml. Sediments with organic soil were found to be less effective in the removal of floodwater organic N, organic C and P, compared to the sediments with calcareous clay loam. Phosphorus exchange rates were dependent on the capacity of the sediment to adsorb or desorb P. Total P exchange rates were in the range of ?1.04 to 0.34 mg P/m² day. Seasonal temperature fluctuations, turbulent vs. quiscent water conditions or exposing the floodwater surface to daylight or dark had very little effect on N and P exchange rates.     

Analysis of the Global Architecture of Hemoglobin A2 by Heme Binding Studies and Molecular Modeling

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A₂ was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5?13 × 10⁷ M^?1 s^?1), local structural rearrangement (0.21?0.23 s^?1), global αδ structural event (0.071?0.098 s^?1), and formation of the Fe–His bond (0.009?0.012 s^?1), respectively. Kinetic differences observed between apohemoglobin A₂ and apohemoglobin A (previously studied) prompted an analysis of the structures of β and δ chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.     

Structure-function relation in hemoglobin Köln (β98 val → met)

Hemoglobin Köln, an unstable hemoglobin resulting from the substitution of normal valine by methionine at FG 5 (β98) is the most commonly encountered unstable hemoglobin. In Hb Köln from a hitherto undetected family, we confirmed earlier observations of heme depletion and high oxygen affinity, with the absence of co-operative interactions. In an effort to elucidate the basis of the altered oxygen equilibria, sedimentation velocity and the kinetics of the reactions of the abnormal hemoglobin with ligands were studied. The results of ultracentrifuge studies indicated that at pH 7 hemoglobin Köln, in the liganded as well as in the deoxy form, existed largely as dimers. The ratio of optical densities at 540 nm and 280 nm indicate that the abnormal β chains were heme depleted. Hb Köln reacted with

approximately 20 times faster than did hemoglobin A (Hb A). However, the corresponding rate constants for O₂ dissociation

are similar for Hb Köln and Hb A. For Hb Köln the two rate constants, l′ and k show little pH or concentration dependence. Thus, the high oxygen affinity of Hb Köln (P_1/2 = 0.2 mm Hg at 10 °C, pH 6.8) arises in part from a larger combination rate constant for the reaction with oxygen. Addition of a 20-fold excess of 2,3-diphosphoglyceric acid did not affect the kinetics of CO-combination. However, in the presence of a sixfold excess of heme, the fast monophasic CO-combination reaction was replaced by a biphasic one. The rate constant of the slow phase was approximately the same as the corresponding rate constant for Hb A. The fast and slow phases were presumably due to the reaction of CO with Hb Köln dimers (α^hβ^o heme-saturated tetramers, respectively. The results of the preeent study are explained in terms of weakened α?β and heme-globin contacts in the mutant.     

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (K_D). Values of K_D ranging from 10^?6m at pH 2.7, 0 °C, to 2 × 10^?10m at pH 7.0, 0 °C, are reported here. Our value for K_D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K_D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.     

Studies on erythrocruorin. IV. Circular dichroism of earthworm erythrocruorin.

Circular dichroism spectra of Lumbricus erythrocruorin in the absence and in the presence of heme ligands have been analyzed under a variety of experimental conditions in view of the peculiarities in ligand binding displayed by this high molecular weight heme protein (M_r = 3 × 10⁶).The undisaociated molecule exists in a “metastable” form with high cooperativity in oxygen binding, which can be converted into a stable form with low co-operativity either by changes in pH or temperature; circular dichroism spectra of oxyerythrocruorin in the Soret region give direct evidence of a local alteration in the heme environment under the conditions which affect co-operativity in oxygen binding of the undissociated molecule. Similar, although more pronounced changes in the same spectral region are observed in the dissociated molecule of M_r = 270,000, which displays low co-operativity in oxygen binding.Deoxygenation is accompanied by an inversion in the double Soret-Cotton effect, which indicates a substantial rearrangement in the heme environment upon removal of the ligand.The double peak in the Soret region found in all erythrocruorin derivatives can be taken as an indication of a distinctive distribution of the aromatic side-chains interacting with the heme chromophore.     

Thermally induced hydrogen exchange processes in small proteins as seen by FTIR spectroscopy

Fourier-transform infrared (FTIR) spectroscopy has been used to study the thermally induced exchange characteristics of those backbone amide protons which persist H-D exchange at ambient conditions in ribonuclease A, in wild type ribonuclease T1 and some of its variants, and in the histone-like protein HBsu. The H-D exchange processes were induced by increasing the thermal energy of the protein solutions in two ways: (i) by linearly increasing the temperature, and (ii) by a temperature jump. To trace the H-D exchange in the proteins, various infrared absorption bands known to be sensitive to H-D exchange were used as specific monitors. Characteristic H-D exchange curves were obtained from which the endpoints (T_H/D) of H-D exchange could be determined. The H-D exchange curves, the T_H/D-values and the phase transition temperatures T_m were used to estimate the structural flexibility and stability of the given proteins. It is suggested that time-resolved FTIR spectroscopy can be used to determine global stability parameters of proteins.     

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability ΔG_U(H₂O) = 7.2 ± 0.25 kcal mol⁻¹ is in good agreement with hydrogen exchange stability ΔG_op. While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.     

pH dependence of conformational fluctuations of the protein backbone

Proton binding equilibria (pK_a values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK_a values measured for individual ionizable residues with NMR spectroscopy are actually population‐weighted averages of the pK_a in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK_a values of surface Glu and Asp residues are affected by pH‐sensitive fluctuations of the backbone between folded and locally unfolded conformations. ¹⁵N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps‐ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub‐global unfolding near carboxylic groups. This study has implications for structure‐based pK_a calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH‐sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co‐operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK_a values of ionizable residues in proteins. Proteins 2014; 82:3132–3143. © 2014 Wiley Periodicals, Inc.