Why Are Proteins Charged? Networks of Charge–Charge Interactions in Proteins Measured by Charge Ladders and Capillary Electrophoresis

Almost all proteins contain charged amino acids. While the function in catalysis or binding of individual charges in the active site can often be identified, it is less clear how to assign function to charges beyond this region. Are they necessary for solubility? For reasons other than solubility? Can manipulating these charges change the properties of proteins? A combination of capillary electrophoresis (CE) and protein charge ladders makes it possible to study the roles of charged residues on the surface of proteins outside the active site. This method involves chemical modification of those residues to generate a large number of derivatives of the protein that differ in charge. CE separates those derivatives into groups with the same number of modified charged groups. By studying the influence of charge on the properties of proteins using charge ladders, it is possible to estimate the net charge and hydrodynamic radius and to infer the role of charged residues in ligand binding and protein folding.     

Simultaneous determination of structural and thermodynamic effects of carbohydrate solutes on the thermal stability of ribonuclease A

This communication describes a new technique for the study of the effects of carbohydrates on the thermal stability of proteins. This approach combines capillary electrophoresis (CE) and protein charge ladders, collections of proteins that differ incrementally in number of chemically modified charged groups, to provide information on both the thermodynamics (i.e., the free energy, DeltaGN-D, of denaturation), and structural changes (i.e., the effective hydrodynamic radius, RH, of proteins in both the native and denatured states) associated with stability. This information, obtained in a single set of electrophoresis experiments, allows a simple microscopic interpretation of the effects of carbohydrate solutes on protein stability. We use this technique to show that the stabilization of ribonuclease A at pH 8.4 by sucrose and fructose can be explained entirely by the contribution these solutes make to the entropy of formation of the protein-solution interface. There is no need, in this case, to refer to quasichemical concepts such as preferential hydration, binding, or exchange of solutes with water at specific sites on the protein to account for the stabilizing effects observed.     

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pK_a values of multiple non‐coordinating residues (predominantly histidine) that involve between 0.42–0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein–cofactor–solvent complex.     

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.     

Dynamic unfolding of a regulatory subunit of cAMP-dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability

Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIalpha) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIalpha by CE in a buffer devoid of cAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligand relative to receptor. This process enabled in-capillary generation of cAMP-stripped RIalpha, which was used to estimate the apparent dissociation constant (Kd) of 0.6 +/- 0.2 microM. A comparison of RIalpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIalpha-cAMP) and without (i.e., cAMP-stripped RIalpha) excess cAMP in the buffer during electromigration. The presence of cAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (DeltaG(U) degrees) of 10.1 +/- 0.5 kcal x mol(+1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal x mol(-1) M(-1). CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.     

Probing electrostatic interactions and structural changes in highly charged protein polyanions by conformer-selective photoelectron spectroscopy

We have recorded the first conformer-selective photoelectron spectra of a protein polyanion in the gas-phase. Bovine cytochrome c protein was studied in 8 different negative charge states ranging from 5- to 12-. Electron binding energies were extracted for all charge states and used as a direct probe of intramolecular Coulomb repulsion. Comparison of experimental results with simulations shows that the experimental outcome can be reproduced with a simple electrostatic model. Energetics are consistent with a structural transition from a folded to an unfolded conformational state of the protein as the number of charges increases. Furthermore, the additional ion-mobility data show that the onset of unfolding can be assigned to charge state 6- where three conformers can be distinguished.     

Size and sequence and the volume change of protein folding

The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance with Le Chatelier's principle, that the unfolded state has a smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔV(u), has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔV(u) using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants that depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long-range coupling intrinsic to the folded state. We also show that the energetics of the protein's conformations can significantly modulate their volumetric properties, providing further insight into protein stability.     

Spermine binding to Parkinson's protein alpha-synuclein and its disease-related A30P and A53T mutants

Aggregation of alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease (PD), is believed to progress through formation of a partially folded intermediate. Using nanoelectrospray ionization (nano-ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for alpha-syn and its disease-related A53T mutant with net charges of -6, -7, and -8. For the other early onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of -6. When bound to spermine near physiologic pH, all three proteins underwent a charge reduction from the favored solution charge state of -10 to a net charge of -6. This charge reduction is accompanied by a dramatic size reduction of about a factor of 2 (cross section of 2600 A2 (-10 charge state) down to 1430 A2 (-6 charge state)). We conclude that spermine increases the aggregation rate of alpha-syn by inducing a collapsed conformation, which then proceeds to form aggregates.     

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

A series of calculations, varying from simple electrostatic to more detailed semi-empirical based molecular dynamics ones, were carried out on charged gas phase ions of the cytochrome c(') dimer. The energetics of differing charge states, charge partitionings, and charge configurations were examined in both the low and high charge regimes. As well, preliminary free energy calculations of dissociation barriers are presented. It is shown that one must always consider distributions of charge configurations, once protein relaxation effects are taken into account, and that no single configuration dominates. All these results also indicate that in the high charge limit, the dissociation of protein complex ions is governed by electrostatic repulsion from the net charges, the consequences of which are enumerated and discussed. There are two main trends deriving from this, namely that charges will move so as to approximately maintain constant surface charge density, and that the lowest barrier to dissociation is the one that produces fragment ions with equal charges. In particular, it is shown that the charge-to-mass ratio of a fragment ion is not the key physical parameter in predicting dissociation products. In fact, from the perspective of the division of total charge, many dissociation pathways reported to be "asymmetric" in the literature should be more properly labelled as "symmetric" or "near-symmetric". The Coulomb repulsion model assumes that the timescale for charge transfer is faster than that for protein structural changes, which in turn is faster than that for complex dissociation.     

Complexes of native ubiquitin and dodecyl sulfate illustrate the nature of hydrophobic and electrostatic interactions in the binding of proteins and surfactants

A previous study, using capillary electrophoresis (CE) [J. Am. Chem. Soc. 2008, 130, 17384-17393], reported that six discrete complexes of ubiquitin (UBI) and sodium dodecyl sulfate (SDS) form at different concentrations of SDS along the pathway to unfolding of UBI in solutions of SDS. One complex (which formed between 0.8 and 1.8 mM SDS) consisted of native UBI associated with approximately 11 molecules of SDS. The current study used CE and (15)N/(13)C-(1)H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to identify residues in folded UBI that associate specifically with SDS at 0.8-1.8 mM SDS, and to correlate these associations with established biophysical and structural properties of this well-characterized protein. The ability of the surface charge and hydrophobicity of folded UBI to affect the association with SDS (at concentrations below the CMC) was studied, using CE, by converting lys-ε-NH(3)(+) to lys-ε-NHCOCH(3) groups. According to CE, the acetylation of lysine residues inhibited the binding of 11 SDS ([SDS] < 2 mM) and decreased the number of complexes of composition UBI-(NHAc)(8)·SDS(n) that formed on the pathway of unfolding of UBI-(NHAc)(8) in SDS. A comparison of (15)N-(1)H HSQC spectra at 0 mM and 1 mM SDS with calculated electrostatic surface potentials of folded UBI (e.g., solutions to the nonlinear Poisson-Boltzmann (PB) equation) suggested, however, that SDS binds preferentially to native UBI at hydrophobic residues that are formally neutral (i.e., Leu and Ile), but that have positive electrostatic surface potential (as predicted from solutions to nonlinear PB equations); SDS did not uniformly interact with residues that have formal positive charge (e.g., Lys or Arg). Cationic functional groups, therefore, promote the binding of SDS to folded UBI because these groups exert long-range effects on the positive electrostatic surface potential (which extend beyond their own van der Waals radii, as predicted from PB theory), and not because cationic groups are necessarily the site of ionic interactions with sulfate groups. Moreover, SDS associated with residues in native UBI without regard to their location in α-helix or β-sheet structure (although residues in hydrogen-bonded loops did not bind SDS). No correlation was observed between the association of an amino acid with SDS and the solvent accessibility of the residue or its rate of amide H/D exchange. This study establishes a few (of perhaps several) factors that control the simultaneous molecular recognition of multiple anionic amphiphiles by a folded cytosolic protein.     

Intrinsic charge ladders of a monoclonal antibody in hydroxypropylcellulose-coated capillaries

Capillary zone electrophoresis (CZE) has been used to resolve the charge heterogeneity of an intact ( approximately 150 kDa) monoclonal IgG antibody (mAb). Although this microheterogeneity can also be observed by isoelectric focusing, CZE allows the net charge of each variant to be measured as a function of pH and other solution conditions. Separation was achieved in both borate and Tris run buffers using capillaries that had been statically coated with hydroxypropylcellulose (HPC). The HPC coating makes inadvertent chromatographic retention of the mAb undetectably small and decreases electroosmotic flow (EOF) to approximately 10(-5) cm(2) V(-1) s(-1), with reasonable stability over dozens of runs under the conditions tested (pH 8.5 and 9.0 for each buffer). We also describe a novel means of measuring small, positive EOF coefficients and larger, negative net mobilities in the same run. This allows determination of accurate electrophoretic mobilities despite variations in EOF. The resolved mAb charge variants (which most likely result from deamidation or partial truncation) constitute what we call an "intrinsic" charge ladder. As with conventional charge ladders formed by deliberate modification of a homogeneous protein, net charge is obtained by extrapolating a plot of electrophoretic mobility versus (assumed) incremental charge difference. At a given pH, the mAb is more negatively charged in borate than in Tris, reflecting specific binding of the B(OH)(4)(-) anion. We also report hydrodynamic radii calculated from the slopes of these plots.     

Energetics of binding and protein unfolding upon amphiphilic drug complexation with a globular protein in different aqueous media

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by zeta-potential, isothermal titration calorimetry (ITC), UV-vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. zeta-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from zeta-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV-vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water (DeltaG(w)(degrees)) and in a hydrophobic environment (DeltaG(hc)(degrees)). Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.     

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.     

Role of opposite charges in protein electrospray ionization mass spectrometry

The conformation dependence of protein spectra recorded by electrospray ionization mass spectrometry (ESI-MS) is an interesting and useful phenomenon, whose origin is still the object of debate. Different mechanisms have been invoked in the attempt to explain the lower charge state of folded versus unfolded protein ions in ESI-MS, such as electrostatic repulsions, solvent accessibility, charge availability, and native-like interactions. In this work we try to subject to direct experimental test the hypothesis that conformation-dependent neutralization of charges with polarity opposite to the net charge of the protein ion could play a critical role in such an effect. We present results of time-of-flight nano-ESI-MS on the peptide angiotensin II, indicating that negative charges of carboxylate groups can contribute to spectra recorded in positive-ion mode when stabilized by favorable electrostatic interactions, which is the central assumption of our hypothesis. Comparison of horse and spermwhale myoglobin (Mb) shows that changing the total number of basic residues within a given three-dimensional structure shifts the charge-state distribution (CSD) of the folded protein in positive-ion mode. This result appears to be in contrast to models in which electrostatic repulsions or availability of charges in the ESI droplets represent the limiting factor for the ionization of folded protein ions in ESI-MS. At the same time, it suggests a role of acidic residues in conformational effects in positive-ion mode. Furthermore, an attempt is made to rationalize those cases in which, in contrast, the main charge state observed in ESI-MS under non-denaturing conditions deviates considerably from the net charge expected on the basis of the amino-acid composition. These cases usually correspond to proteins with quite balanced content in basic and acidic residues, suggesting that this might be a factor influencing their charging behavior in ESI-MS. Experiments on mutants of ribonuclease Sa (RNase Sa) reveal that progressively reducing the excess of acidic residues, replacing them by lysine, causes almost no shift in the spectrum of the folded protein in negative-ion mode. Analogously, variants with an excess of three or five basic residues give similar spectra in positive-ion mode. These results indicate a lower limit to the extent of ionization observable by ESI-MS (6- or 8+ in the case of RNase Sa in water). Below such limit of net charge, changes in the relative amount of ionizable side chains do not affect the qualitative features of the observed CSDs. A progressive loss of signal intensity caused by the mutations in negative-ion mode suggests that low charge states might also be counterselected, even within the m/z range theoretically accessible to the instrument.     

Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.     

Interpreting conformational effects in protein nano-ESI-MS spectra

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is employed here to describe equilibrium protein conformational transitions and to analyze the influence of instrumental settings, pH, and solvent surface tension on the charge-state distributions (CSD). A first set of experiments shows that high flow rates of N₂ as curtain gas can induce unfolding of cytochrome c (cyt c) and myoglobin (Mb), under conditions in which the stability of the native protein structure has already been reduced by acidification. However, it is possible to identify conditions under which the instrumental settings are not limiting factors for the conformational stability of the protein inside ESI droplets. Under such conditions, equilibrium unfolding transitions described by ESI-MS are comparable with those obtained by other established biophysical methods. Experiments with the very stable proteins ubiquitin (Ubq) and lysozyme (Lyz) enable testing of the influence of extreme pH changes on the ESI process, uncoupled from acid-induced unfolding. When HCl is used for acidification, Ubq and Lyz mass spectra do not change between pH~7 and pH 2.2, indicating that the CSD is highly characteristic of a given protein conformation and not directly affected by even large pH changes. Use of formic or acetic acid for acidification of Ubq solutions results in major spectral changes that can be interpreted in terms of protein unfolding as a result of the increased hydrophobicity of the solvent. On the other hand, Lyz, cyt c, and Mb enable direct comparison of protein CSD (corresponding to either the folded or the unfolded protein) in HCl or acetic acid solutions at low pH. The values of surface tension for these solutions differ significantly. Confirming indications already present in the literature, we observe very similar CSD under these solvent conditions for several proteins in either compact or disordered conformations. The same is true for comparison between water and water–acetic acid for folded cyt c and Lyz. Thus, protein CSD from water–acetic solutions do not seem to be limited by the low surface tension of acetic acid as previously suggested. This result could reflect a general lack of dependence of protein CSD on the surface tension of the solvent. However, it is also possible that the effect of acetic acid on the precursor ESI droplets is smaller than generally assumed.     

pH-induced structural transitions of caseins

Caseins are relatively small (molecular mass approximately 20 kDa), unstructured milk proteins of which the main components are referred to as alpha(s)-, beta- and kappa-casein. All three components lack a compact folded conformation, which can be ascribed to a combination of their low overall hydrophobicity and high net charge. Structural transitions of the three caseins in response to variation of pH were investigated using fluorescence and circular dichroism (CD) spectroscopy. Tryptophan emission parameters (intensity and wavelength of emission maximum) and CD spectra showed that at neutral and alkaline pH the caseins exist predominantly in random coil conformation. As the solvent is made acidic the added protons compensate the negative charges on the caseins and reduce the repulsion between like charged residues, allowing the casein chains to fold. At the pI (pH 4-5), the net charge on the protein tends to zero and the protein should approach its maximally structured state. Below pI, the uncompensated charges and their interactions reappear, resulting in slackening of the compact structure and formation of a partially unfolded intermediate. These conclusions were borne out by the biphasic pH-dependence of the fluorescence emission parameters of Trp as well as of ANS incubated with the caseins. Measurement of the efficiency of energy transfer between Trp (donor) and ANS (acceptor) and of the CD spectra of caseins as functions of pH were also consistent with this scenario.     

Hydrodynamic radius ladders of proteins

We introduce hydrodynamic radius ladders of proteins as a new tool to isolate and measure the role of hydrodynamic size on transport properties of proteins. Radius ladders are collections of derivatives of a protein that differ incrementally in number of polyethylene glycol (PEG) chains grafted to their surface. The addition of these chains causes the hydrodynamic size of the protein to increase. Capillary electrophoresis (CE) separates these derivatives into individual peaks or "rungs" of a ladder composed of proteins that have the same number of PEG chains, and provides a way to measure the values of hydrodynamic radius of proteins that constitute the rungs of the ladder. We demonstrate the utility of this approach by measuring the partitioning of radius ladders into polymer hydrogels. The combination of radius ladders and CE produces a large amount of internally consistent data on hydrodynamic size. This technique will have applicability to the study of the role of hydrodynamic size on transport.     

Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.