Change In Ionization State Research Articles

Double Shells of the Planetary Nebula NGC 7009 Minor Axis

We analyzed the minor-axis spectra of the elliptical planetary nebula (PN) NGC 7009 observed with Keck HIRES with a 0.″862 × 10″ slit placed at about 7.″5 and 10″ away from the center and a 0.″862 × 14″ slit at the center. The mean densities derived from the integrated [S ii] 6716/6731 Å fluxes along the Keck HIRES slit length indicate a density range of 103.7–104.1 cm−3, while the local densities derived from the slit spectral images show a large local density variation of about 102.8–104.6 cm−3: local densities vary more substantially than values integrated over the line of sight. The expansion rates of the main and outer shells obtained by [S ii] are about 21.7 and 30.0 km s−1, respectively. The kinematic results of the [S ii] spectral lines correspond to the outermost regions of the two shells and are not representative of the whole PN but are closely related to the other emission lines observed in the shell gas. We conclude that the density contrast leads to the formation of the inner shell, while the change in ionization state leads to the formation of the outer shell. We suggest that the inner main and outer shells result from two successive major ejections. The physical conditions of the central star must have been different when these shells first formed.

The Correlated Variations of Absorption Lines and Quasar Continuum

Abstract Using the quasar spectra from the Sloan Digital Sky Survey and with variable C iv absorption line systems, we measure the Si iv and N v absorption line systems. We obtain 50 variable Si iv absorption line systems and 39 variable N v absorption line systems. We find that the variations in most of the C iv, Si iv, and N v absorption lines are correlated with the changes in quasar continuum. In addition, a significant portion of the variable absorption lines are the consistent variations of multiple systems with large velocity separations. Therefore, the variations of the C iv, Si iv, and N v absorption lines could be mainly driven by the changes in quasar radiations, which cause changes in ionization states or column densities of absorbing gas. We also find that the variable C iv, Si iv, and N v absorption line systems can be divided into low-ionization systems and high-ionization systems. The former positively responds to the changes in the quasar’s brightness, and the later is the oppositive case.

The Sloan Digital Sky Survey Reverberation Mapping Project: Systematic Investigations of Short-timescale C IV Broad Absorption Line Variability

Abstract We systematically investigate short-timescale (<10 day rest-frame) C iv broad absorption-line (BAL) variability to constrain quasar-wind properties and provide insights into BAL-variability mechanisms in quasars. We employ data taken by the Sloan Digital Sky Survey Reverberation Mapping project, as the rapid cadence of these observations provides a novel opportunity to probe BAL variability on shorter rest-frame timescales than have previously been explored. In a sample of 27 quasars with a median of 58 spectral epochs per quasar, we have identified 15 quasars ( %), 19 of 37 C iv BAL troughs ( %), and 54 of 1460 epoch pairs (3.7% ± 0.5%) that exhibit significant C iv BAL equivalent-width variability on timescales of less than 10 days in the quasar rest frame. These frequencies indicate that such variability is common among quasars and BALs, though somewhat rare among epoch pairs. Thus, models describing BALs and their behavior must account for variability on timescales down to less than a day in the quasar rest frame. We also examine a variety of spectral characteristics and find that, in some cases, BAL variability is best described by ionization-state changes, while other cases are more consistent with changes in covering fraction or column density. We adopt a simple model to constrain the density and radial distance of two outflows appearing to vary by ionization-state changes, yielding outflow density lower limits consistent with previous work.

SAMPL6 challenge results from [Formula: see text] predictions based on a general Gaussian process model.

A variety of fields would benefit from accurate [Formula: see text] predictions, especially drug design due to the effect a change in ionization state can have on a molecule's physiochemical properties. Participants in the recent SAMPL6 blind challenge were asked to submit predictions for microscopic and macroscopic [Formula: see text]s of 24 drug like small molecules. We recently built a general model for predicting [Formula: see text]s using a Gaussian process regression trained using physical and chemical features of each ionizable group. Our pipeline takes a molecular graph and uses the OpenEye Toolkits to calculate features describing the removal of a proton. These features are fed into a Scikit-learn Gaussian process to predict microscopic [Formula: see text]s which are then used to analytically determine macroscopic [Formula: see text]s. Our Gaussian process is trained on a set of 2700 macroscopic [Formula: see text]s from monoprotic and select diprotic molecules. Here, we share our results for microscopic and macroscopic predictions in the SAMPL6 challenge. Overall, we ranked in the middle of the pack compared to other participants, but our fairly good agreement with experiment is still promising considering the challenge molecules are chemically diverse and often polyprotic while our training set is predominately monoprotic. Of particular importance to us when building this model was to include an uncertainty estimate based on the chemistry of the molecule that would reflect the likely accuracy of our prediction. Our model reports large uncertainties for the molecules that appear to have chemistry outside our domain of applicability, along with good agreement in quantile-quantile plots, indicating it can predict its own accuracy. The challenge highlighted a variety of means to improve our model, including adding more polyprotic molecules to our training set and more carefully considering what functional groups we do or do not identify as ionizable.

Spectroscopic Observations of the Outflowing Wind in the Lensed Quasar SDSS J1001+5027∗

Abstract We performed spectroscopic observations of the small-separation lensed quasar SDSS J1001+5027, whose images have an angular separation , and placed constraints on the physical properties of gas clouds in the vicinity of the quasar (i.e., in the outflowing wind launched from the accretion disk). The two cylinders of sight to the two lensed images go through the same region of the outflowing wind and they become fully separated with no overlap at a very large distance from the source (∼330 pc). We discovered a clear difference in the profile of the C iv broad absorption line (BAL) detected in the two lensed images in two observing epochs. Because the kinematic components in the BAL profile do not vary in concert, the observed variations cannot be reproduced by a simple change of ionization state. If the variability is due to gas motion around the background source (i.e., the continuum source), the corresponding rotational velocity is ≥ 18,000 km s−1, and their distance from the source is pc assuming Keplerian motion. Among three Mg ii and three C iv NAL systems that we detected in the spectra, only the Mg ii system at = 0.8716 shows a hint of variability in its Mg i profile on a rest-frame timescale of 191 days and an obvious velocity shear between the sightlines whose physical separation is ∼7 kpc. We interpret this as the result of motion of a cosmologically intervening absorber, perhaps located in a foreground galaxy.

Continuum Electrostatics Approaches to Calculating pKas and Ems in Proteins.

Proteins change their charge state through protonation and redox reactions as well as through binding charged ligands. The free energy of these reactions is dominated by solvation and electrostatic energies and modulated by protein conformational relaxation in response to the ionization state changes. Although computational methods for calculating these interactions can provide very powerful tools for predicting protein charge states, they include several critical approximations of which users should be aware. This chapter discusses the strengths, weaknesses, and approximations of popular computational methods for predicting charge states and understanding the underlying electrostatic interactions. The goal of this chapter is to inform users about applications and potential caveats of these methods as well as outline directions for future theoretical and computational research.

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3kcal/mol to set the enzyme into a catalytically competent form.

Searching for the trigger of the active galactic nucleus quasi-periodic oscillation: 8 years of RE J1034+396

RE J1034+396 is one of the most extreme Narrow-line Seyfert 1s detected thus far, showing the only quasi-periodic oscillation (QPO) reliably detected in an Active Galactic Nucleus (AGN). Comparison with similar spectral and timing properties observed in the black hole X-ray binary (BHB) GRS 1915+105 suggests that RE J1034+396 is a super-Eddington accretor. A more complete understanding of the behaviour of RE J1034+396 can therefore lead to a unification of the accretion physics between such extreme AGN and super-Eddington BHBs. Here we report on our latest XMM-Newton observations of RE J1034+396, which no longer show the QPO, indicating that this source shows a non-stationary power-spectrum. We use spectral and temporal analysis across all five XMM-Newton observations of the source to probe the evolution of the object. The combination of the shape of the fractional variability with energy and the inferred velocity of absorbing material in the line-of-sight rules out an absorption-only method of creating the QPO. Instead the periodically changing absorption may be produced by the QPO causing a change in ionization state. We extend our analysis by including the covariance spectra which give much better signal to noise than an rms spectrum. These reveal a new aspect of the QPO, which is that there is also a small contribution from a soft component which is hotter than the soft excess seen in the mean spectrum. Folding the lightcurve on the QPO period shows that this component lags behind the hard X-rays. If this is due to re-processing then the lag corresponding to a light travel time across 30 R_g. Some of the remaining observations have similar energy spectra and covariance spectra, but none of them show a significant QPO, so we conclude that none of these features are the trigger for the appearance of the QPO in this object.

Application of the Gaussian dielectric boundary in Zap to the prediction of protein pKa values

The results of two rounds of blind pK(a) predictions for ionizable residues in staphylococcal nuclease using OpenEye's legacy protein pK(a) prediction program based on the Zap Poisson-Boltzmann solver were submitted to the 2009 prediction challenge organized by the Protein pK(a) Cooperative and first round predictions were discussed at the corresponding June 2009 Telluride conference. To better understand these results, 21 additional sets of predictions were performed with the same program, varying the internal dielectric, reference pK(a), partial charge set, and dielectric boundary. The internal dielectric (ε(p)) and dielectric boundary were the two most important factors contributing to the quality of the predictions. Although the lowest overall errors were observed with a molecular dielectric boundary at ε(p) = 8, predictions using a smooth Gaussian dielectric boundary performed almost as well at lower ε(p) values because the Gaussian boundary implicitly accounts for a significant level of solvent penetration. Improved pK(a) predictions with the Gaussian boundary methodology will require better prediction and modeling of structural changes due to changes in ionization state, perhaps without resorting to the more exhaustive sampling of conformational states used by other recent continuum methods.

PH-Dependent Transport of Pemetrexed by Breast Cancer Resistance Protein

Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment.

Statistics and Physical Origins of pK and Ionization State Changes upon Protein-Ligand Binding

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4–8, the number of ionizable residues that experience substantial pK changes (ΔpK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.

Statistics and Physical Origins of pK and Ionization State Changes Upon Protein-Ligand Binding

Statistics and Physical Origins of pK and Ionization State Changes Upon Protein-Ligand Binding

SPITZERSPECTRAL MAPPING OF SUPERNOVA REMNANT CASSIOPEIA A

We present the global distribution of fine structure infrared line emission in the Cassiopeia A supernova remnant using data from the Spitzer Space Telescope's Infrared Spectrograph. We identify emission from ejecta materials in the interior, prior to their encounter with the reverse shock, as well as from the post-shock bright ring. The global electron density increases by >~100 at the shock to ~10^4 cm^-3, providing evidence for strong radiative cooling. There is also a dramatic change in ionization state at the shock, with the fading of emission from low ionization interior species like [SiII], giving way to [SIV] and, at even further distances, high-energy X-rays from hydrogenic silicon. Two compact, crescent-shaped clumps with highly enhanced neon abundance are arranged symmetrically around the central neutron star. These neon crescents are very closely aligned with the "kick" direction of the compact object from the remnant's expansion center, tracing a new axis of explosion asymmetry. They indicate that much of the apparent macroscopic elemental mixing may arise from different compositional layers of ejecta now passing through the reverse shock along different directions.

Results of Monitoring the Dramatically Variable CivMini–Broad Absorption Line System in the Quasar HS 1603+3820

We present six new and two previously published high-resolution spectra of the quasar HS 1603+3820 (z_em = 2.542) taken over an interval of 4.2 years (1.2 years in the quasar rest frame). The observations were made with the High-Dispersion Spectrograph on the Subaru telescope and Medium-Resolution Spectrograph on the Hobby-Eberly Telescope. The purpose was to study the narrow absorption lines (NALs). We use time variability as well as coverage fraction analysis to separate intrinsic absorption lines, which are physically related to the quasar, from intervening absorption lines. By fitting models to the line profiles, we derive the parameters of the respective absorbers as a function of time. Only the mini-BAL system at z_abs ~ 2.43 (v_off ~ 9,500 km/s) shows both partial coverage and time variability, although two NAL systems possibly show evidence of partial coverage. We find that all the troughs of the mini-BAL system vary in concert and its total equivalent width variations resemble those of the coverage fraction. However, no other correlations are seen between the variations of different model parameters. Thus, the observed variations cannot be reproduced by a simple change of ionization state nor by motion of a homogeneous parcel of gas across the cylinder of sight. We propose that the observed variations are a result of rapid continuum fluctuations, coupled with coverage fraction fluctuations caused by a clumpy screen of variable optical depth located between the continuum source and the mini-BAL gas. An alternative explanation is that the observed partial coverage signature is the result of scattering of continuum photons around the absorber, thus the equivalent width of the mini-BAL can vary as the intensity of the scattered continuum changes.

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

BackgroundEnzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.ResultsAs model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-β-cyclodextrin (MβCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.ConclusionThese results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

Calculations of pH-Dependent Binding of Proteins to Biological Membranes

Binding of proteins to membranes is often accompanied by titration of ionizable residues and is, therefore, dependent on pH. We present a theoretical treatment and computational approach for predicting absolute, pH-dependent membrane binding free energies. The standard free energy of binding, DeltaG, is defined as -RTln(P(b)/P(f)), where P(b) and P(f) are the amounts of bound and free protein. The apparent pK(a) of binding is the pH value at which P(b) and P(f) are equal. Proteins bind to the membrane in the pH range where DeltaG is negative. The components of the binding free energy are (a) the free energy cost of ionization state changes (DeltaG(ion)), (b) the effective energy of transfer from solvent to the membrane surface, (c) the translational/rotational entropy cost of binding, and (d) an ideal entropy term that depends on the relative volume of the bound and free state and therefore depends on lipid concentration. Calculation of the first term requires determination of pK(a) values in solvent and on the membrane surface. All energies required by the method are obtained from molecular dynamics trajectories on an implicit membrane (IMM1-GC). The method is tested on pentalysine and the helical peptide VEEKS, derived from the membrane-binding domain of phosphocholine cytidylyltransferase. The agreement between the measured and the calculated free energies of binding of pentalysine is good. The extent of membrane binding of VEEKS is, however, underestimated compared to experiment. Calculations of the interaction energy between two VEEKS helices on the membrane suggest that the discrepancy is mainly due to the neglect of protein-protein interactions on the membrane surface.

Time‐Variable Complex Metal Absorption Lines in the Quasar HS 1603+3820

We present a new spectrum of the quasar HS 1603+3820 taken 1.28 yr (0.36 yr in the quasar rest frame) after a previous observation with Subaru+HDS. The new spectrum enables us to search for time variability as an identifier of intrinsic narrow absorption lines (NALs). This quasar shows a rich complex of C IV NALs within 60,000 km s-1 of the emission redshift. On the basis of covering factor analysis, Misawa et al. found that the C IV NAL system at zabs = 2.42-2.45 (system A, at a shift velocity of vsh = 8300-10,600 km s-1 relative to the quasar) was intrinsic to the quasar. With our new spectrum, we perform time variability analysis, as well as covering factor analysis, to separate intrinsic NALs from intervening NALs for eight C IV systems. Only system A, which was identified as an intrinsic system in the earlier paper by Misawa et al., shows a strong variation in line strength (Wobs ≈ 10.4 → 19.1 Å). We speculate that a broad absorption line (BAL) could be forming in this quasar (i.e., many narrower lines will blend together to make a BAL profile). We illustrate the plausibility of this suggestion with the help of a simulation in which we vary the column densities and covering factors of the NAL complex. Under the assumption that a change of ionization state causes the variability, a lower limit can be placed on the electron density (ne ≳ 3 × 104 cm-3) and an upper limit on the distance from the continuum source (r ≤ 6 kpc). On the other hand, if the motion of clumpy gas causes the variability (a more likely scenario), the crossing velocity and the distance from the continuum source are estimated to be vcross > 8000 km s-1 and r < 3 pc. In this case, the absorber does not intercept any flux from the broad emission line region, but only flux from the UV continuum source. If we adopt the dynamical model of Murray et al., we can obtain a much more strict constraint on the distance of the gas parcel from the continuum source, r < 0.2 pc.

Combining Conformational Flexibility and Continuum Electrostatics for Calculating pK as in Proteins

Protein stability and function relies on residues being in their appropriate ionization states at physiological pH. In situ residue pK as also provides a sensitive measure of the local protein environment. Multiconformation continuum electrostatics (MCCE) combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side chain ionization and conformation. The response of protein to charges is incorporated both in the protein dielectric constant ( ɛ prot) of four and by explicit conformational changes. The pK a of 166 residues in 12 proteins was determined. The root mean square error is 0.83 pH units, and >90% have errors of <1 pH units whereas only 3% have errors >2 pH units. Similar results are found with crystal and solution structures, showing that the method's explicit conformational sampling reduces sensitivity to the initial structure. The outcome also changes little with protein dielectric constant ( ɛ prot 4–20). Multiconformation continuum electrostatics titrations show coupling of conformational flexibility and changes in ionization state. Examples are provided where ionizable side chain position (protein G), Asn orientation (lysozyme), His tautomer distribution (RNase A), and phosphate ion binding (RNase A and H) change with pH. Disallowing these motions changes the calculated pK a.

Volatile buffers can override the "pH memory" of subtilisin catalysis in organic media.

The protonation state and activity of enzymes in low-water media are affected by the aqueous pH before drying ("pH memory"). However, both protonation and activity will change if buffer ions can be removed as volatile or organic-extractable weak acids or bases. With NH4OOCH buffers, in which both ions can be removed, pH memory disappears completely for subtilisin-catalyzed transesterification in hexane. Only weak pH memory is found with buffers having one volatile component, NH4-phosphate and NaOOCH. The changes in ionization state result from proton exchanges like Protein-COO-NH4+ --> Protein-COOH + NH3 (g) and Protein-NH3+HCOO- --> Protein-NH2 + HOOCH (g). An equivalent, complementary picture is that net charges on the protein and buffer ions must remain equal and opposite. With NaOOCH buffers, loss of some HCOO- ions gives a more negative net charge on the protein, balanced by the excess Na+. With NH4-phosphate buffers, loss of NH3 gives protein with a more positive net charge. The resulting catalytic activities were high and low, respectively, similar to those after drying from Na-phosphate buffers of optimal (8.5) and acid pH. All of the above effects have been demonstrated for both covalently immobilized subtilisin and the lyophilized free enzyme. Subtilisin lyophilized from NH4OOCH buffers gave pH approximately 4 after redissolution in water, probably because removal of HCOO- counterions remains incomplete. The resulting catalytic activity was low. The effects are discussed in relation to the possible locations, in low-dielectric media, of the positive charge that balances the net negative catalytic triad in active subtilisin.